Multiple coordinate controls contribute to a balanced expression of ribulose-1,5-bisphosphate carboxylase/oxygenase subunits in rye leaves

In the leaves of rye (Secale cereale L.), control mechanisms acting at multiple molecular levels contribute to a coordinate expression of the subunit polypeptides of ribulose-1,5-bisphosphate carboxylase. The relevance and hierarchy of the different control steps were evaluated by comparing the time courses of changes in levels of translatable mRNA, rates of in vivo amino acid incorporation, and the turnover of subunit polypeptides after selective interference with translation at either cytoplasmic 80S ribosomes, or at the 70S ribosomes of the chloroplast, by compartment-specific inhibitors, or by the use of 70S-ribosome-deficient leaves. The latter were generated by growing the plants at a non-permissive elevated temperature of 32 degrees C. The rates of synthesis of the two ribulose-1,5-bisphosphate carboxylase subunits were most rapidly adapted to each other by translational controls. Within 0.5-2.5 h after selective inhibition of the synthesis of either subunit, that of the other subunit made in the unaffected compartment also declined by more than 90% without any marked change in its mRNA. After prolonged inhibition (24 h) of either cytoplasmic or chloroplast protein synthesis, the levels of mRNAs for both subunits were greatly diminished. In rye, the mRNA levels for both subunits changed under all experimental conditions tested in a closely parallel manner and appeared to be always maintained in a balanced, fairly constant ratio by strong coordinate controls. Even 70S-ribosome-deficient leaves contained mRNAs for both the small and the large subunits, although only in small amounts. The mRNAs for both subunits were also markedly further decreased in 70S-ribosome-deficient leaves after application of an inhibitor of cytoplasmic translation. MDMP [2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide], suggesting that the suppression of the large subunit mRNA in the plastids was not mediated through feedback effects of accumulating unassembled large subunits. Coordinate controls at both the mRNA and the translational level require a bidirectional exchange of regulatory signals between chloroplast and cytoplasm. However, these controls were not absolutely restrictive and allowed low rates of uncoupled synthesis of either large or small subunits. Large subunits made in the presence of MDMP were stable over 24 h. However, unassembled small subunits synthesized in 70S-ribosome-deficient leaves were degraded with a half-time of 10.5 h, in contrast to their behavior after integration into the holoprotein in normal leaves, where no turnover was detected. The proteolytic removal of surplus free small subunits is regarded as a final post-translational fine-tuning step to establish a balanced subunit stoichiometry in leaves.     

Prairie grasses as hosts of the northern corn rootworm (Coleoptera: Chrysomelidae)

We evaluated 27 prairie grass species thought to be among those dominant 200 yr ago in the northern midwest as larval hosts of the northern corn rootworm, Diabrotica barberi Smith and Lawrence. Maize (Zea mays L.), spring wheat (Triticum aestivum L.), and grain sorghum (Sorghum bicolor L.) were included as controls for a total of 30 species. Twenty pots of each species were planted in a randomized complete block design. Each pot was infested 5 wk later with 20 neonate northern corn rootworm larvae. Two pots within each species and block were assigned an extraction date of 7 or 14 d after infestation. The remaining two pots from each block were used to monitor adult emergence. The percentage of larvae recovered, change in larval head capsule width, and change in average dry weights varied significantly among the grass species. The highest percentage of larvae was recovered from slender wheatgrass, Elymus trachycaulus (Link), and this was significantly greater than the percentage recovered from all other species including maize for the 14-d sample date. Several additional species were also relatively good hosts, in that the percentage of larvae recovered from these species was not significantly different from maize. The average dry weight of larvae recovered was significantly greater for larvae recovered from maize than for larvae recovered from all other species except slender wheatgrass, when the two samples dates were combined. Overall, adults were produced from only 6 of the 28 species evaluated, and no analysis was performed because of the low numbers. The results of this study are discussed in relation to the potential of alternate hosts of northern corn rootworm to serve as a bridge to survival on transgenic maize.     

Breeding soybeans for the tropics capable of nodulating effectively with indigenousRhizobium spp.

Summary Most soybean varieties fail to nodulate effectively in tropical soils unless inoculated with a competitive strain ofRhizobium japonicum. Developing countries in the tropics, with few exceptions, lack inoculant industries to produce and distribute viable inoculants to small farmers and extension programs to teach them to use inoculant. Several soybean genotypes have been identified that nodulate effectively with many strains of the cowpea inoculation group which is ubiquitous in tropical soils of Africa. Soybean genotypes that nodulate and grow well without inoculant application are called promiscuous. Methodologies for incorporation of the promiscuity character into high-yielding backgrounds are discussed.Supported in part by grant 05-0560 from United Nations Development Program to the International Institute of Tropical Agriculture.     

Resistance of cold-hardened winter rye leaves (Secale cereale L.) to photo-oxidative stress

Catalase and photosystem II (PSII) were strongly inactivated during exposure to 4 °C and moderate light in 22 °C-grown non-hardened leaves (NHL) of winter rye (Secale cereale L.), but highly resistant to photo-inactivation at low temperature in 4 °C-grown cold-hardened leaves (CHL). Resistance of CHL to chilling-induced photo-inactivation of catalase and PSII depended partially on more efficient de novo synthesis at 4 °C and partially on improved protection. Lower rates of chloroplast-mediated inactivation of catalase in vitro indicated that less reactive oxygen was released by chloroplasts from CHL than by chloroplasts from NHL. The contents of xanthophyll cycle carotenoids, α-tocopherol, ascorbate, glutathione, the activities of superoxide dismutase and glutathione reductase, and the tolerance against paraquat-induced photo-oxidative damage were greatly increased in CHL, relative to NHL. Zeaxanthin-related thermal energy dissipation was only of minor importance for paraquat-tolerance and protection of catalase in CHL. When CHL were transferred to a higher temperature of 22 °C the increased resistance to photo-inactivation of catalase and PSII and the increased paraquat-tolerance were largely lost within 3 d, whereas most non-enzymic and enzymic antioxidants retained higher levels than in NHL. The decline of resistance to photodamage during dehardening was not related to concomitant changes of antioxidants or antioxidative enzymes.     

Mode of interference of chlorosis-inducing herbicides with peroxisomal enzyme activities

In rye leaves ( Secale cereale L. cv. Petkus "Kustro") bleached in the presence of the chlorosis-inducing herbicides aminotriazole, haloxidine, San 6706 or difunone in white light of 54.2 W m^-2 (5000 lx), catalase activity was very low. In addition, the activities of glycolate oxidase and hydroxypyruvate reductase were strongly diminished in treatments with San 6706 and difunone. The lowering of the peroxisomal enzyme activities was observed in red, but not in blue light and did not occur after treatment with the non-bleaching pyridazinone derivative San 9785. The deficiencies of the peroxisomal enzymes did not appear to be involved in the initiation of the chlorosis. Instead they are probably produced as secondary consequences of the bleaching. Low peroxisomal enzyme activities were also obtained without herbicide treatment by growing the leaves in an atmosphere of 2% O₂ and 3% CO₂, but in this case were not accompanied by an increased sensitivity of the Chl to photooxidative bleaching. The peroxisomal enzymes reached as high activities as in untreated controls when the herbicide-treated leaves were grown at a low light intensity of 0.106 W m^-2 (10 lx). After transfer of herbicide-treated leaves grown under 0.106 W m^-2 to 306 W m^-2 (30 000 lx), catalase was strongly inactivated, even at 0°C. In treatments with San 6706 and difunone the increase of the activities of glycolate oxidase and hydroxypyruvate reductase was either stopped, remaining unchanged, or the enzymes were slightly inactivated after exposure to 306 W m^-2 (30 000 lx). The observations suggest that the inactivation of peroxisomal enzymes results from photooxidative events in the chloroplasts.     

Metabolism of activated oxygen in detached wheat and rye leaves and its relevance to the initiation of senescence

Pollen grains of Plumbago zeylanica L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and organelle content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a long (approx. 30 m) projection, which wraps around and lies within embayments of the vegetative nucleus. This cell contains numerous mitochondria, up to two plastids and, infrequently, microbodies. It is characterized by a larger volume and surface area and contains a larger nucleus than the other sperm cell. The second sperm cell (S_ua) is linked by plasmodesmata with the S_vn, but is unassociated with the vegetative nucleus. It is smaller and lacks a cellular projection. The S_ua contains relatively few mitochondria, but numerous (up to 46) plastids and more microbodies than the other sperm. The degree of dimorphism in their content of heritable cytoplasmic organelles must at fertilization result in nearly unidirectional transmission of sperm plastids into just one of the two female reproductive cells, and preferential transmission of sperm mitochondria into the other.Abbreviations S_ua sperm cell unassociated with the vegetative nucleus - S_vn sperm cell physically associated with the vegetative nucleus 1=Russell and Cass (1981)     

Myelin-associated glycoprotein modulates expression and phosphorylation of neuronal cytoskeletal elements and their associated kinases

Decreased phosphorylation of neurofilaments in mice lacking myelin-associated glycoprotein (MAG) was shown to be associated with decreased activities of extracellular-signal regulated kinases (ERK1/2) and cyclin-dependent kinase-5 (cdk5). These in vivo changes could be caused directly by the absence of a MAG-mediated signaling pathway or secondary to a general disruption of the Schwann cell-axon junction that prevents signaling by other molecules. Therefore, in vitro experimental paradigms of MAG interaction with neurons were used to determine if MAG directly influences expression and phosphorylation of cytoskeletal proteins and their associated kinases. COS-7 cells stably transfected with MAG or with empty vector were co-cultured with primary dorsal root ganglion (DRG) neurons. Total amounts of the middle molecular weight neurofilament subunit (NF-M), microtubule-associated protein 1B (MAP1B), MAP2, and tau were up-regulated significantly in DRG neurons in the presence of MAG. There was also increased expression of phosphorylated high molecular weight neurofilament subunit (NF-H), NF-M, and MAP1B. Additionally, in similar in vitro paradigms, total and phosphorylated NF-M were increased significantly in PC12 neurons co-cultured with MAG-expressing COS cells or treated with a soluble MAG Fc-chimera. The increased expression of phosphorylated cytoskeletal proteins in the presence of MAG in vitro was associated with increased activities of ERK 1/2 and cdk5. We propose that interaction of MAG with an axonal receptor(s) induces a signal transduction cascade that regulates expression of cytoskeletal proteins and their phosphorylation by these proline-directed protein kinases.     

Photoinactivation of Catalase Occurs under Both High- and Low-Temperature Stress Conditions and Accompanies Photoinhibition of Photosystem II

Severe photoinactivation of catalase (EC 1.11.1.6) and a decline of variable fluorescence (F_v), indicating photoinhibition of photosynthesis, were observed as rapid and specific symptoms in leaves exposed to a high heat-shock temperature of 40°C as well as in leaves exposed to low chilling temperatures in white light of only moderately high photosynthetic photon flux density of 520 μE m⁻² s⁻¹. Other parameters, such as peroxidase (EC 1.11.1.7), glycolate oxidase (EC 1.1.3.1), glutathione reductase (EC 1.6.4.2), or the chlorophyll content, were hardly affected under these conditions. At a compatible temperature of 22°C, the applied light intensity did not induce severe photoinactivations. In darkness, exposures to high or low temperatures did not affect catalase levels. Also, decline of F_v in light was not related to temperature sensitivity in darkness. The effective low-temperature ranges inducing photoinactivation of catalase differed significantly for chilling-tolerant and chilling-sensitive plants. In leaves of rye (Secale cereale L.) and pea (Pisum sativum L.), photoinactivation occurred only below 15°C, whereas inactivation occurred at 15°C in cucumber (Cucumis sativus L.) and maize (Zea mays L.). The behavior of F_v was similar, but the difference between chilling-sensitive and chilling-tolerant plants was less striking. Whereas the catalase polypeptide, although photoinactivated, was not cleaved at 0 to 4°C, the D1 protein of photosystem II was greatly degraded during the low-temperature treatment of rye leaves in light. Rye leaves did not exhibit symptoms of any major general photodamage, even when they were totally depleted of catalase after photoinactivation at 0 to 4°C, and catalase recovered rapidly at normal temperature. In cucumber leaves, the decline of catalase after exposures to bright light at 0 to 4°C was accompanied by bleaching of chlorophyll, and the recovery observed at 25°C was slow and required several days. Similar to the D1 protein of photosystem II, catalase differs greatly from other proteins by its inactivation and high turnover in light. Inasmuch as catalase and D1 protein levels depend on continuous repair synthesis, preferential and rapid declines are generally to be expected in light whenever translation is suppressed by stress actions, such as heat or chilling, and recovery will reflect the repair capacity of the plants.     

Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation.