cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase

A cDNA clone encoding the cytosolic ascorbate peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While ascorbate peroxidase shares limited overall homology with other peroxidases, significant homology with all known peroxidases was found in the vicinity of the putative active site.     

Molecular cloning of a cytosolic ascorbate peroxidase cDNA from cell cultures of sweetpotato and its expression in response to stress

A cDNA encoding a cytosolic ascorbate peroxidase (APX), swAPX1 , was isolated from cell cultures of sweetpotato (Ipomoea batatas) by cDNA library screening, and its expression in the context of various environmental stresses was investigated. swAPX1 contains an ORF of 250 amino acids (27.5 kDa) encoding a protein with a pI value of 5.32. The swAPX1 ORF does not code for a transit peptide, suggesting that the product is a cytosolic isoform. RNA blot analysis showed that swAPX1 gene is expressed in cultured cells and mature leaves, but not in stems, non-storage or storage roots of sweetpotato. The level of swAPX1 RNA progressively increased during cell growth in suspension cultures. In leaf tissues, the gene responded differentially to various abiotic stresses, as revealed by RT-PCR analysis. swAPX1 was highly induced in leaves by wounding, and treatment with methyl viologen (50 M), hydrogen peroxide (440 mM), abscisic acid (ABA; 100 M) or exposure to high temperature (37°C). In addition, the gene was strongly induced in the leaves following inoculation with a bacterial pathogen (Pectobacterium chrysanthemi). These results indicate that swAPX1 may be involved in hydrogen peroxide-detoxification and thus help to overcome the oxidative stress induced by abiotic and biotic stresses.Communicated by G. Jürgens     

Characterization of populations of rapid-cycling Brassica rapa L. selected for differential waterlogging tolerance

By challenging a heterogenous population of plants (rapid-cyclingBrassica rapa L.) with waterlogging stress, we selected plantswhich differed in their response to root zone hypoxia. Theseindividuals were placed into ‘tolerant’ and ‘sensitive’populations based on foliage colour after waterlogging and werethen mass-pollinated and re-selected over seven generationsto produce the stable populations described. To assess responsesto root zone hypoxia in the selected populations, plants weregrown for 1 week after germination under normal watering conditionsand then subjected to waterlogging stress for up to 8 d. Undercontrol conditions, no differences were found between the tolerantand sensitive populations in any of the parameters studied.Chlorophyll concentrations in the tolerant population were significantlygreater than the concentrations in the sensitive populationwhen plants had been waterlogged. A similar stress-specificdifference was found in root and shoot dry matter accrual. Assoil redox values (and hence, available oxygen) decreased, anincrease in soluble carbohydrates and starch occurred in theleaves of waterlogged plants. Changes in soluble carbohydrateswere noted as early as 12 h after waterlogging in the sensitiveplants, and starch concentrations were significantly higherfor this population 24 h after waterlogging. Under waterloggedconditions, activities of alcohol dehydrogenase (ADH) and pyruvatedecarboxylase (PDC) increased, phosphoglucomutase and malatedehydrogenase decreased, and malic enzyme and glucose 6-phosphatedehydrogenase did not change. The sensitive population exceededthe tolerant population in activities of ADH and PDC after 18and 48 h of waterlogging, respectively. The results demonstratethat stress-specific differences in population responses towaterlogging can be achieved through recurrent selection. Key words: Waterlogging, carbohydrates, selection, hypoxia, Brassica rapa     

cDNA cloning,characterization and expression of an endosperm-specific barley peroxidase

A barley peroxidase (BP 1) of pI ca. 8.5 and M _r 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from a cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed maximal expression 15 days after flowering, and high levels were found only in the endosperm. BP 1 was not expressed in the leaves.     

cDNA cloning and differential gene expression of three catalases in pumpkin

Three cDNA clones (cat1, cat2, cat3) for catalase (EC 1.11.1.6) were isolated from a cDNA library of pumpkin (Cucurbita sp.) cotyledons. In northern blotting using the cDNA-specific probe, the cat1 mRNA levels were high in seeds and early seedlings of pumpkin. The expression pattern of cat1 was similar to that of malate synthase, a characteristic enzyme of glyoxysomes. These data suggest that cat1 might encode a catalase associated with glyoxysomal functions. Furthermore, immunocytochemical analysis using cat1-specific anti-peptide antibody directly showed that cat1 encoding catalase is located in glyoxysomes. The cat2 mRNA was present at high levels in green cotyledons, mature leaf, stem and green hypocotyl of light-grown pumpkin plant, and correlated with chlorophyll content in the tissues. The tissue-specific expression of cat2 had a strong resemblance to that of glycolate oxidase, a characteristic enzyme of leaf peroxisomes. During germination of pumpkin seeds, cat2 mRNA levels increased in response to light, although the increase in cat2 mRNA by light was less than that of glycolate oxidase. cat3 mRNA was abundant in green cotyledons, etiolated cotyledons, green hypocotyl and root, but not in young leaf. cat3 mRNA expression was not dependent on light, but was constitutive in mature tissues. Interestingly, cat1 mRNA levels increased during senescence of pumpkin cotyledons, whereas cat2 and cat3 mRNAs disappeared during senescence, suggesting that cat1 encoding catalase may be involved in the senescence process. Thus, in pumpkin, three catalase genes are differentially regulated and may exhibit different functions.     

Molecular cloning, expression and mapping analysis of a novel cytosolic ascorbate peroxidase gene from tomato.

Ascorbate peroxidase (APX, EC 1.11.1.11) plays a major role in H(2)O(2)-scavenging in plants and can help to avoid reactive oxygen species (ROS) damage. A new cytosolic APX gene was cloned from tomato (designated LecAPX2) by RACE-PCR. The full-length cDNA of LecAPX2 contained a complete open reading frame (ORF) of 753 bp, which encoding 250 amino acid residues. Homology analysis of LecAPX2 showed a 94% identity with potato cAPX gene and 92% identity with another tomato cAPX gene (APX20), the deduced amino acid showed 88% homology with APX20 protein and 75-92% identity with cAPX from other plants such as potato, tobacco, broccoli, spinach, pea, rice, etc. LecAPX2 revealed the existence of a haem peroxidase and plant APX family signatures. Northern blot analysis showed that LecAPX2 was constitutively expressed in root, stem, leaf, flower and fruit of tomato, whereas the expression levels were different. LecAPX2 was mapped to 6-A using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii.     

Pollination and embryo development in Brassica rapa L. in microgravity

Plant reproduction under spaceflight conditions has been problematic in the past. In order to determine what aspect of reproductive development is affected by microgravity, we studied pollination and embryo development in Brassica rapa L. during 16 d in microgravity on the space shuttle (STS-87). Brassica is self-incompatible and requires mechanical transfer of pollen. Short-duration access to microgravity during parabolic flights on the KC-135A aircraft was used initially to confirm that equal numbers of pollen grains could be collected and transferred in the absence of gravity. Brassica was grown in the Plant Growth Facility flight hardware as follows. Three chambers each contained six plants that were 13 d old at launch. As these plants flowered, thin colored tape was used to indicate the date of hand pollination, resulting in silique populations aged 8-15 d postpollination at the end of the 16-d mission. The remaining three chambers contained dry seeds that germinated on orbit to produce 14-d-old plants just beginning to flower at the time of landing. Pollen produced by these plants had comparable viability (93%) with that produced in the 2-d-delayed ground control. Matched-age siliques yielded embryos of equivalent developmental stage in the spaceflight and ground control treatments. Carbohydrate and protein storage reserves in the embryos, assessed by cytochemical localization, were also comparable. In the spaceflight material, growth and development by embryos rescued from siliques 15 d after pollination lagged behind the ground controls by 12 d; however, in the subsequent generation, no differences between the two treatments were found. The results demonstrate that while no stage of reproductive development in Brassica is absolutely dependent upon gravity, lower embryo quality may result following development in microgravity.     

Genomic Markers Associated with Cold-Hardiness in Brassica rapa L.

Molecular Biology - Brassica rapa L. is a valuable and widespread species, but its cultivation in risk farming areas requires high-quality cold-hardy varieties to be developed. Mechanisms of the...     

Dynamics of storage reserve deposition during Brassica rapa L. pollen and seed development in microgravity

Pollen and seeds share a developmental sequence characterized by intense metabolic activity during reserve deposition before drying to a cryptobiotic form. Neither pollen nor seed development has been well studied in the absence of gravity, despite the importance of these structures in supporting future long-duration manned habitation away from Earth. Using immature seeds (3-15 d postpollination) of Brassica rapa L. cv. Astroplants produced on the STS-87 flight of the space shuttle Columbia, we compared the progress of storage reserve deposition in cotyledon cells during early stages of seed development. Brassica pollen development was studied in flowers produced on plants grown entirely in microgravity on the Mir space station and fixed while on orbit. Cytochemical localization of storage reserves showed differences in starch accumulation between spaceflight and ground control plants in interior layers of the developing seed coat as early as 9 d after pollination. At this age, the embryo is in the cotyledon elongation stage, and there are numerous starch grains in the cotyledon cells in both flight and ground control seeds. In the spaceflight seeds, starch was retained after this stage, while starch grains decreased in size in the ground control seeds. Large and well-developed protein bodies were observed in cotyledon cells of ground control seeds at 15 d postpollination, but their development was delayed in the seeds produced during spaceflight. Like the developing cotyledonary tissues, cells of the anther wall and filaments from the spaceflight plants contained numerous large starch grains, while these were rarely seen in the ground controls. The tapetum remained swollen and persisted to a later developmental stage in the spaceflight plants than in the ground controls, even though most pollen grains appeared normal. These developmental markers indicate that Brassica seeds and pollen produced in microgravity were physiologically younger than those produced in 1 g. We hypothesize that microgravity limits mixing of the gaseous microenvironments inside the closed tissues and that the resulting gas composition surrounding the seeds and pollen retards their development.     

Isolation and characterization of microsatellites in Brassica rapa L

We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)(15) and (GT)(15) oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F(2) population from a cross of doubled-haploid lines DH27 x G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.     

cDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli

The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastidlocalized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Identification of a male sterile gene Ms in Brassica rapa L.

Molecular Breeding - Flowering time is an important agronomic trait, which is of great significance to the plant growth process. Salicylic acid (SA) is a key hormone that regulates plant growth and...