Phytochrome control of plastid mRNA in mustard (Sinapis alba L.)

The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with ³²P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.Abbreviations LS large subunit - RuBP ribulose-1,5-bisphosphate - ptDNA plastid DNA     

Effects of red clover (Trifolium pratense) green manure and compost soil amendments on wild mustard (Brassica kaber) growth and incidence of disease

Using a soil bioassay technique, seedling growth and incidence of disease of wild mustard (Brassica kaber) and sweet corn (Zea mays) were assessed in soil from field plots that received either of two treatments: incorporated red clover (Trifolium pratense) residue plus application of compost (`amended soil'), or application of ammonium nitrate fertilizer (`unamended soil'). Soils were analyzed for percent moisture, dissolved organic carbon, conductivity, phenolics, and nutrient content. A trend toward greater incidence of Pythium spp. infection of wild mustard seedlings grown in amended soil was observed during the first 40 days after incorporation (DAI) of red clover and compost, with significant differences ( = 0.05) at two out of four sampling dates in 1997, and four out of four sampling dates in 1998. Incidence of Pythium infection was 10–70% greater in the amended soil treatment during that period. Asymptomatic wild mustard seedlings grown in amended soil were also on average 2.5 cm shorter ( = 0.05) at 5 DAI than those grown in unamended soil in one year out of two. Concentration of phenolic compounds in soil solution was weakly correlated with decreased shoot and root growth (r = 0.50, 0.28, respectively) and increased incidence of disease (r = 0.48) in wild mustard seedlings in one year out of two. Dissolved organic carbon concentration was weakly correlated with increased disease in wild mustard seedlings in both years (r = 0.51, 0.33, respectively). Growth of corn seedlings did not differ between the two soil treatments, suggesting that red clover green manure and compost may selectively reduce density and competitive ability of wild mustard in the field. Bioassay results corresponded well with emergence and shoot weight results from a related field study, indicating that this technique may be useful for screening potential soil treatments prior to field studies.     

Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag⁺/Cs₂SO₄ density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag⁺/Cs₂SO₄ density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm³ and a heavy shoulder at density 1,703 g/cm³ are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag⁺ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag⁺/Cs₂SO₄ gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag⁺/Cs₂SO₄ satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag⁺/Cs₂SO₄ gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag⁺/Cs₂SO₄ gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.     

Redundancy modulation of nuclear DNA sequences in Dasypyrum villosum

In order to assess fluid domains in the genome of Dasypyrum villosum, Feulgen/DNA cytophotometric determinations and molecular and cytological DNA-DNA hybridization experiments were carried out in resting embryos and developing seedlings from yellow and brown caryopses belonging to different populations. The cytophotometric data showed that the basic amount of nuclear DNA is, on average, 12% higher in 2-day-old seedlings from yellow caryopses as compared to those from brown caryopses. It increases in each individual during seed germination, to a higher extent in seedlings from yellow caryopses than in those from brown caryopses. DNA content also differs up to 13% between plants within a caryopsis-colour group and up to 40% between populations. Dot-blot hybridization of a 396-bp D. villosum-specific DNA repeat to genomic DNA extracted from embryos in dry seeds, or from seedlings belonging to single progenies of plants from different populations, confirmed the cytophotometric results. The redundancy in the genome of sequences hybridizing to the 396-bp element differs significantly both between populations and between plant progenies within a population. During seed germination these sequences are the more amplified the less they are redundant in the genome of resting embryos, and amplification occurs to a significantly-greater extent in seedlings from yellow caryopses than in those from brown caryopses. ³H-labelled 396-bp sequences hybridize at or near the telomeres of most chromsome pairs though only to the shorter of the two subtelocentric pairs. The hybridization level is higher in seedlings from yellow caryopses that in those from brown caryopses, and a linear correlation exists between the number of silver grains counted over the labelled regions of each chromosome pair in the two groups of seedlings. Possible control mechanisms of the observed changes in the nuclear genome, and the role of these changes in developmental pregulation and environmental adaptation, are discussed.     

Induction of amylase in mustard seedlings by phytochrome

Summary In the cotyledons of mustard seedlings (Sinapis alba L.) amylase activity can be induced by phytochrome. In the dark amylase activity remains low. Gibberellic acid (GA₃) does not stimulate an increase of amylase activity in this system. Inhibitors of RNA and protein synthesis strongly inhibit the increase of amylase activity mediated by phytochrome. In gel electrophoresis amylase from mustard seedlings reveals 3 bands. The electrophoretic pattern is the same for extracts from dark-grown and from irradiated seedlings. When mustard amylases were incubated with starch the pattern of products was similar to that produced by commercially available barley -amylase and not similar to that produced by Bacillus subtilis -amylase.     

Studies on five species of Pythium parasitic on mustard and cabbage in India

Diseased seedlings and roots of cabbage and mustard of Khurpatal (Nainital), were surveyed for zoosporic plant pathogens. Pythium aphanidermatum and P. debaryanum were isolated from cabbage and P. middletonii, P. spinosum and P. undulatum were isolated from mustard. Pathogenicity tests conducted in the glasshouse indicated that P. debaryanum and P. spinosum are the most virulent pathogens of cabbage and mustard respectively.     

Cytological localization of fast renaturing and satellite DNA sequences inVicia faba

Summary DNA sequences reassociating within a Cot value of 1.8×10^–1 and those producing a light satellite in a CsCl density gradient were isolated fromVicia faba DNA and hybridizedin situ on squashes of roots of the same species. Silver grains were seen to be scattered over both the interphase nuclei and the metaphase chromosomes after hybridization with fast renaturing DNA sequences, indicating these are fairly regularly interspersed in theV. faba genome. Clustered labeling occurred after hybridization with satellite DNA sequences, indicating these are clustered in the genome. The localization of satellite DNA in chromosomes appeared to correspond closely to the position of the bright bands detectable after staining with quinacrine mustard. After hybridization with both DNA probes, labeling intensity over the nuclei of meristematic cells was higher than that over the nuclei of differentiating and/or differentiated cells. These results are discussed in relation to the structure of the cell nucleus, the mechanism of quinacrine banding and to previous data suggesting underrepresentation of nuclear repeated DNA sequences in differentiatingV. faba root cells.     

Utilizing RNA/DNA hybridization to directly quantify mRNA levels in microbial fermentation samples

mRNA quantification has become a research hotspot. Quantitative real-time RT-PCR is a popular method but is known to lack precision. To rapidly monitor the kinetics of mRNA levels for the control of microbial fermentation processes, we developed an SYBR Green I-based universal method to directly quantify mRNA from fermentation samples. After total RNA was extracted, the mRNA was hybridized and protected by a longer DNA oligonucleotide. The probe length determined the strength of signal amplification. S1 nuclease and RNase A were used to remove excess probe, single-stranded RNA, and mis-matched RNA/DNA hybrids. Finally, the perfect-matched RNA/DNA hybrid was quantified by SYBR Green I dye. The conditions of liquid hybridization and enzyme digestion were systemically optimized. The kinetic tendency of phzC mRNA levels during phenazine-1-carboxylic acid fermentation was consistent with the results from MB hybridization in our previous report. The detection of mRNA levels of ten genes in Pseudomonas sp. M18G proved that this method is universal and feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.     

Comparison of the action of light on ribosomal RNA synthesis in cabbage and mustard seedlings

We have compared the action of light on ribosomal RNA synthesis in mustard and cabbage seedlings, two of the most frequently used systems for the studies of anthocyanin synthesis. The level of RNA (both t-RNA and r-RNA) “stored” in mustard dry seeds is much lower than in cabbage dry seeds. The kinetics of RNA synthesis in cabbage and mustard seedlings exposed to light are very different: In cabbage seedlings, light produces no apparent stimulation of cytoplasmic r-RNA synthesis, while it does increase plastid r-RNA synthesis. On the other hand, in mustard seedlings, light promotes both cytoplasmic and plastid ribosomal RNA synthesis. Streptomycin, which inhibits chlorophyll formation and chloroplast development while having no effect (mustard) or enhancing (cabbage) anthocyanin synthesis in these two systems, is in both cases an effective inhibitor of plastid r-RNA synthesis, but not of cytoplasmic r-RNA synthesis.     

T-DNA presence and opine production in tumors of Picea abies (L.) Karst induced by Agrobacterium tumefaciens A281

The hypervirulent Agrobacterium tumefaciens strain A281 formed frequent tumors (31%) on Picea abies (Norway spruce), an economically important tree species in Swedish forests. Three-month-old seedlings were inoculated and tumors were established that grew hormone-independently in culture. Tumors contained agropine and mannopine/mannopinic acid as determined by acid pH paper electrophoresis. In addition, DNA hybridization studies showed that the DNA from these tumor lines contained sequences homologous to Ti plasmid T-DNA, whereas wild-type spruce seedling DNA did not. These results suggest that Agrobacterium vectors can be used for gene transfer into this important forest species.     

Molecular cloning of a pea mRNA encoding an early light induced,nuclear coded chloroplast protein

Summary cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2–4 h after onset of illumination in five day old, etiolated pea seedlings. The cDNA library was constructed from poly(A)⁺-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA established the basis of our screening procedure. Colony hybridization experiments were performed with³²P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of M_r 24 000. After posttranslational importin vitro, the processed product of M_r 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2–4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones.     

Effect of organic amendments on seedling infection of rice caused byRhizoctonia solani

Summary Soils amended with organic amendments such as rice chaff, mustard cake, neem cake, saw dust and farm yard manure significantly reduced the seedling infection of rice owing toRhizoctonia solani. Significant increase in shoot growth was noticed in soils amended with organic amendments while reduction in root growth of rice seedlings was also observed owing to the addition of organic amendments such as sunflower cake, mustard cake, coconut cake and farmyard manure.     

5S-RNA genes of barley are located on the second chromosome

Summary The genes coding for 5S RNA in barley were cloned, sequenced, and their cluster was assigned to chromosome 2 using wheat-barley chromosome addition lines. High-resolution gel-electrophoresis of DNA and subsequent hybridization revealed new details of the organization of 5S DNA both in wheat and barley. The in situ hybridization of the cloned 5S gene with triploid endosperm nuclei also suggests that these genes are located in a single locus.On leave from: Department of Plant Breeding and Genetics, College of Agriculture, Orissa University of Agriculture & Technology, Bhubaneswar-751003, India     

The size of poly(A)-containing RNAs in Drosophila melanogaster embryos

The size range of poly(A)-containing RNA from Drosophila melanogaster embryos has been estimated by hybridization with ³H-labeled poly(U) and subsequent fractionation on sucrose gradients. The median size of nuclear poly(A)-containing RNA is about 30 S (6000 nucleotides), and the median size of cytoplasmic poly(A)-containing RNA is about 17 S (1800 nucleotides). The relationship of these sizes to messenger RNA needed to code for protein and to the length of DNA contained in a chromomere is discussed.Research grant support was provided by NIH (6M35558; HD-00266) and NSF (GB-30600).     

Expression of two cloned mRNA sequences during development and germination of spores of the sensitive fern,Onoclea sensibilis L.

A library of complementary DNA (cDNA) clones has been prepared from poly(A)⁺RNA of spores of the sensitive fern, Onoclea sensibilis L. By differential hybridization with labeled probes made to poly(A)⁺ RNA of spores, gametophytes and leaves, two spore-specific clones (pOSS68 and pOSS194) were selected and characterized. Northern blot analysis showed that RNA sequences homologous to the two cDNA clones first appear in the post-meiotic spore and increase in abundance during spore maturity. Both RNA sequences decay during photoinduced germination of the spores and do not reappear in the gametophytes. In spores imbibed in the dark under conditions which do not favor germination, no significant decrease in pOSS194-mRNA abundance is noted. In contrast, the decrease in pOSS68 mRNA in dark-imbibed spores parallels that observed in photoinduced spores. The predicted amino-acid sequence of pOSS194 has a striking similarity to the early light-inducible proteins expressed during the greening of etiolated pea and barley seedlings, whereas that of pOSS68 shows some homology to proteins encoded by late-embryogenesis-abundant mRNAs of angiosperm embryos.Abbreviations bp base pairs - cDNA complementary DNA - ds double-stranded - ELIP early light-inducible proteins - LEA late embryogenesis abundant - nt nucleotide - ss single stranded This work was partially supported by a NASA grant (NAGW-901) and by an allocation from the Research Challenge Investigators' Fund of the Ohio State University to V.R. Thanks are due to Mr. Clayton L. Rugh for sequencing our clones and to Dr. Paul A. Fuerst for help in the computer search of sequence alignments.     

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

Summary Near-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)⁺RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [³²P]-labelled cDNA prepared from poly(A)⁺RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.