The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and nonacclimated canola leaves.
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Infrared video thermography was used to observe ice nucleation temperatures, patterns of ice formation, and freezing rates in nonacclimated and cold acclimated leaves of a spring (cv Quest) and a winter (cv Express) canola (Brassica napus). Distinctly different freezing patterns were observed, and the effect of water content, sugars, and soluble proteins on the freezing process was characterized. When freezing was initiated at a warm subzero temperature, ice growth rapidly spread throughout nonacclimated leaves. In contrast, acclimated leaves initiated freezing in a horseshoe pattern beginning at the uppermost edge followed by a slow progression of ice formation across the leaf. However, when acclimated leaves, either previously killed by a slow freeze (2 degrees C h(-1)) or by direct submersion in liquid nitrogen, were refrozen their freezing pattern was similar to nonacclimated leaves. A novel technique was developed using filter paper strips to determine the effects of both sugars and proteins on the rate of freezing of cell extracts. Cell sap from nonacclimated leaves froze 3-fold faster than extracts from acclimated leaves. The rate of freezing in leaves was strongly dependent upon the osmotic potential of the leaves. Simple sugars had a much greater effect on freezing rate than proteins. Nonacclimated leaves containing high water content did not supercool as much as acclimated leaves. Additionally, wetted leaves did not supercool as much as nonwetted leaves. As expected, cell solutes depressed the nucleation temperature of leaves. The use of infrared thermography has revealed that the freezing process in plants is a complex process, reminding us that many aspects of freezing tolerance occur at a whole plant level involving aspects of plant structure and metabolites rather than just the expression of specific genes alone. (+info)
AtSGP1, AtSGP2 and MAP4K alpha are nucleolar plant proteins that can complement fission yeast mutants lacking a functional SIN pathway.
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In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is signalled via the septation initiation network (SIN) involving several protein kinases and a GTPase. Arabidopsis thaliana and Brassica napus proteins homologous to fission yeast spg1p (AtSGP1, AtSGP2), cdc7p (AtMAP3K epsilon 1, AtMAP3K epsilon 2, BnMAP3K epsilon 1) and sid1p (AtMAP4K alpha 1, AtMAP4K alpha 2, BnMAP4K alpha 2) exhibit a significant similarity. The plant proteins AtSGP1/2 and BnMAP4K alpha 2 are able to complement the S. pombe mutant proteins spg1-B8 and sid1-239, respectively and to induce mutisepta when overexpressed in wild-type yeast. Yeast two-hybrid assays demonstrated interactions both between plant proteins and between plant and yeast proteins of the SIN pathway. However, the primary structure of the proteins as well as the partial complementation of yeast mutants indicates that plant homologous proteins and their yeast counterparts have diverged during evolution. Real-time RT-PCR studies demonstrated plant SIN-related gene expression in all organs tested and a co-expression pattern during the cell cycle, with a higher accumulation at G(2)-M. During interphase, the plant SIN-related proteins were found to co-localise predominantly in the nucleolus of the plant cells, as shown by fusions to green fluorescent protein. These data suggest the existence of a plant SIN-related pathway. (+info)
Changes in gene expression in canola roots induced by ACC-deaminase-containing plant-growth-promoting bacteria.
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The technique of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) was used to study changes in gene expression over time in canola roots treated with the 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant-growth-promoting bacterium Enterobacter cloacae UW4 and to compare the changes with those in a mutant of E. cloacae UW4 in which the ACC deaminase structural gene acdS was replaced by homologous recombination with acdS with an intentional knockout containing a tetracycline resistance gene. Genes that were either up- or down-regulated over a three-day period in canola plants treated with wild-type or mutant bacteria were isolated, cloned, and sequenced; all appeared to have high homology with Arabidopsis thaliana genes. The upregulated genes included a cell division cycle protein 48 homolog and a eukaryotic translation initiation factor 3 subunit 7 gene homolog. The downregulated genes included one encoding a glycine-rich RNA binding protein with a function in RNA processing or binding during ethylene-induced stress, which is expressed only in roots, and another gene thought to be involved in a defense signaling pathway. All RAP-PCR results were verified using Northern blotting. These data, indicate that roots isolated from canola seeds treated with the ACC deaminase-producing E. cloacae UW4 upregulate genes involved in cell division and proliferation but down-regulate stress genes. This data is in agreement with a model in which ACC deaminase-containing plant-growth-promoting bacteria reduce plant stress and induce root elongation and proliferation in plants, largely by lowering ethylene levels. (+info)
Sulphur supply and infection with Pyrenopeziza brassicae influence L-cysteine desulphydrase activity in Brassica napus L.
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Different field surveys have shown that sulphur (S) fertilization can increase the resistance of agricultural crops against fungal pathogens. The mechanisms of this sulphur-induced resistance (SIR) are, however, not yet known. Volatile S compounds are thought to play an important role because H(2)S is toxic to fungi. A field experiment was conducted to analyse the influence of S fertilization and the activity of H(2)S-releasing enzymes on fungal infections. Two levels of N and S fertilizers and two varieties of oilseed rape were investigated with respect to their potential to release H(2)S by the enzymatic activity of L-cysteine desulphydrase (LCD) and O-acetyl-L-serine(thiol)lyase (OAS-TL). LCD releases H(2)S during cysteine degradation, while OAS-TL consumes H(2)S during cysteine synthesis and free H(2)S is only released in a side reaction. All plots of the field trial showed an infection with Pyrenopeziza brassicae and leaf disc samples were taken from visibly infected leaf areas and apparently uninfected areas to investigate the reaction to the infection in relation to the treatments. Different S fractions and the activities of LCD and OAS-TL were measured to evaluate the potential to release H(2)S in relation to S nutrition and fungal infection. S fertilization significantly increased the contents of total S, sulphate, organic S, cysteine, and glutathione in the plants, but decreased LCD activity. Infection with P. brassicae increased cysteine and glutathione contents, as well as the activity of LCD. Therefore crops were able to react to a fungal infection with a greater potential to release H(2)S, which is reflected by an increasing LCD activity with fungal infection. (+info)
Isolation and characterization of a serine/threonine protein kinase SOS2 gene from Brassica napus.
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A full-length cDNA of a new serine/threonine (Ser/Thr) protein kinase gene, designated as BnSOS2 (GenBank Acc. No.AY310413), was cloned from Brassica napus by rapid amplification of cDNA ends (RACE). The full-length cDNA of BnSOS2 was 1779 bp and contained a 1539-bp open reading frame encoding a protein of 512 amino acids. Homology analysis shows that BnSOS2 strongly resembles other Ser/Thr protein kinase genes, and that its putative protein belongs to a typical Ser/Thr kinase family. Northern blot analysis reveals that BnSOS2 is salt-inducible. Our results indicate that BnSOS2 is a new member of the plant SOS2 gene family, which may play an important role in salt tolerance of plants. (+info)
Embryo-specific reduction of ADP-Glc pyrophosphorylase leads to an inhibition of starch synthesis and a delay in oil accumulation in developing seeds of oilseed rape.
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In oil-storing Brassica napus (rape) seeds, starch deposition occurs only transiently in the early stages of development, and starch is absent from mature seeds. This work investigates the influence of a reduction of ADP-Glc pyrophosphorylase (AGPase) on storage metabolism in these seeds. To manipulate the activity of AGPase in a seed-specific manner, a cDNA encoding the small subunit of AGPase was expressed in the sense or antisense orientation under the control of an embryo-specific thioesterase promoter. Lines were selected showing an embryo-specific decrease in AGPase due to antisense and cosuppression at different stages of development. At early developmental stages (25 days after flowering), a 50% decrease in AGPase activity was accompanied by similar decreases in starch content and the rate of starch synthesis measured by injecting (14)C-Suc into seeds in planta. In parallel to inhibition of starch synthesis, the level of ADP-Glc decreased, whereas Glc 1-phosphate levels increased, providing biochemical evidence that inhibition of starch synthesis was due to repression of AGPase. At 25 days after flowering, repression of starch synthesis also led to a decrease in the rate of (14)C-Suc degradation and its further metabolism via other metabolic pathways. This was not accompanied by an increase in the levels of soluble sugars, indicating that Suc import was inhibited in parallel. Flux through glycolysis, the activities of hexokinase, and inorganic pyrophosphate-dependent phosphofructokinase, and the adenylate energy state (ATP to ADP ratio) of the transgenic seeds decreased, indicating inhibition of glycolysis and respiration compared to wild type. This was accompanied by a marked decrease in the rate of storage lipid (triacylglycerol) synthesis and in the fatty acid content of seeds. In mature seeds, glycolytic enzyme activities, metabolite levels, and ATP levels remained unchanged, and the fatty acid content was only marginally lower compared to wild type, indicating that the influence of AGPase on carbon metabolism and oil accumulation was largely compensated for in the later stages of seed development. Results indicate that AGPase exerts high control over starch synthesis at early stages of seed development where it is involved in establishing the sink activity of the embryo and the onset of oil accumulation. (+info)
Spatially structured population dynamics in feral oilseed rape.
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We studied the population dynamics of feral oilseed rape (Brassica napus) for 10 years (1993-2002) in 3658 adjacent permanent 100 m quadrats in the verges of the M25 motorway around London, UK. The aim was to determine the relative importance of different factors affecting the observed temporal patterns of population dynamics and their spatial correlations. A wide range of population dynamics was observed (downward or upward trends, cycles, local extinctions and recolonizations), but overall the populations were not self-replacing (lambda < 1). Many quadrats remained unoccupied throughout the study period, but a few were occupied at high densities for all 10 years. Most quadrats showed transient oilseed rape populations, lasting 1-4 years. There were strong spatial patterns in mean population density, associated with soil conditions and the successional age of the plant community dominating the verge, and these large-scale spatial patterns were highly consistent from year to year. The importance of seed spilled from trucks in transit to the processing plant at Erith in Kent was confirmed: rape populations were significantly higher on the 'to Erith' verge than the 'from Erith' verge (overall mean 2.83-fold greater stem density). Quadrats in which lambda > 1 were much more frequent in the 'to Erith' verge, indicating that seed immigration can give the spurious impression of self-replacing population dynamics in time-series analysis. There was little evidence of a pervasive Moran effect, and climatic forcing did not produce widespread large-scale synchrony in population dynamics for the motorway as a whole; just 23% of quadrats had significant rank correlations with the mean time-series. There was, however, significant local spatial synchrony of population dynamics, apparently associated with soil disturbance and seed input. This study draws attention to the possibility that different processes may impose population synchrony at different scales. We hypothesize that synchrony in this system is driven by at least three processes: small-scale, local forcing caused by soil disturbance, intermediate-scale forcing as a result of seed input, and large-scale climatic forcing (e.g. winter rainfall) that affects the motorway as a whole. (+info)
The capacity of green oilseeds to utilize photosynthesis to drive biosynthetic processes.
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Seeds of many plant species are green during embryogenesis. To directly assess the influence of light on the physiological status of green oilseeds in planta, Brassica napus and soybean (Glycine max) seeds were rapidly dissected from plants growing in the light or dark. The activation state of malate dehydrogenase, which reflects reduced thioredoxin and NADP/NADPH ratios, was found to be as high in seeds exposed to light as in leaves and to decrease in the dark. Rubisco was highly activated (carbamylated) in both light and dark, most likely reflecting high seed CO(2) concentrations. Activities of Rubisco and phosphoribulokinase were sufficient to account for significant refixation of CO(2) produced during B. napus oil biosynthesis. To determine the influence of light on oil synthesis in planta, siliques on intact plants in full sunlight or detached siliques fed (3)H(2)O were partly covered with aluminum foil. Seeds from light and dark sections were analyzed, and fatty acid accumulation was found to be higher in seeds exposed to light than seeds from dark sections. The spectrum of light filtering through silique walls and the pigment composition of developing B. napus embryos were determined. In addition to a low chlorophyll a/b ratio, the carotenoid pigments of seeds can provide additional capture of the green light that filters through siliques. Together, these results demonstrate that even the low level of light reaching seeds plays a substantial role in activating light-regulated enzymes, increasing fatty acid synthesis, and potentially powering refixation of CO(2). (+info)