The effect of far-red light on the appearance of the radish ribulose 1,5-bisphosphate carboxylase, its subunits and the mRNA for the small subunit has been studied. The immunological analysis of the accumulation of holoenzyme and small subunit showed that there was no pool of free small subunit either in the etiolated seedlings or in the far-red light illuminated seedlings. The precursor to the small subunit has been identified by immunoprecipitation of the in vitro translation products directed by poly(A)-containing RNA of radish cotyledons. The irradiation of radish cotyledons with far-red light led to the apparent increase of the level of the translatable mRNA for the small subunit.
The yield of chlorophyll a variable fluorescence from leaves of grapevine, bean, barley, spinach, citrus (Citrus myrtifolia) and mangrove (Avicennia marina) remained unaffected by salt treatment declining only when excessive ion accumulation led to losses of cell turgor pressure. Salt treatment was found to accelerate the rate of fluorescence quenching after maximal fluorescence yield was attained in barley, spinach, citrus and mangrove, species which accumulate both sodium and chloride into their leaves, but not in grapevine or bean, species which accumulate chloride but exclude sodium from their leaves. Stimulation of fluorescence quenching in intact chloroplasts and thylakoids isolated from spinach was induced upon addition of sodium chloride, but not sorbitol. Energy-dependent fluorescence quenching associated with establishment of a pH gradient appears to be stimulated by sodium ion in isolated chloroplasts and thylakoids. In intact leaves stimulation of fluorescence quenching is more likely a consequence of accelerated photochemical reoxidation of Q, the stable electron acceptor of photosystem II.
Methyl viologen-dependent nitrite reductase (EC 1.7.7.1) (NiR) was purified about 1780-fold with a yield of 3.5% from etiolated bean shoots with a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Butyl-Toyopearl chromatography, ferredoxin-Sepharose affinity chromatography and Ultrogel AcA44 gel filtration. The purified enzyme was apparently homogeneous as shown by polyacrylamide disc gel electrophoresis with a specific activity of 53.4 units/mg protein. The molecular weight of the enzyme was estimated to be 100 kilodaltons by gel filtration. Subunit analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded two protein bands with a large subunit molecular weight of 64 kilodaltons and a small subunit molecular weight of 35 kilodaltons. The purified enzyme could be stored at −20°C for several weeks without any loss of activity in the presence of 10% glycerol and 10 mM ß-mercaptoethanol.
UDP-glucose: 1,3-1,4 ß-glucan synthase was solubilized from red beet (Beta vulgaris L.) root microsomes by treatment with 1% digitonin at 30°C. The enzyme shows unusual stability for a solubilized glucan synthase, with full activity retention after storage for 1 week at 4°C.
Plasma membrane ATPase partially purified from radish seedlings (Raphanus sativum L.) (2.4–3.5 μmol Pi min−1 mg−1 protein) has been reconstituted in proteoliposomes by the cholate-dialysis technique. Proteoliposomes are able to acidify their internal volume in the presence of Mg:ATP. Mg:ATP-dependent proton pumping is prevented by N,N′-dicyclohexylcarbodiimide (DCCD) and by vanadate at the same concentrations which are effective on the phosphohydrolyzing activity of the plasma membrane ATPase.
We demonstrate continued nitrogenase activity in isolated bacteroids of Rhizobium strain ANU289 from nodules of siratro (Macroptilium atropurpureum) and the non-legume Parasponia rigida for periods up to 240 min with maximum activities maintained at 52 nmol ethylene produced h−1 mg−1 protein. Both legume and non-legume bacteroids showed a broader oxygen tolerance in the presence of the nodule supernatant. Isolated Parasponia-derived bacteroids exhibited an optimum nitrogenase activity at about 0.1% O2 in the gas phase, while siratro bacteroids had a slightly higher O2 optimum. These findings illustrate that (i) Rhizobium bacteroids isolated from a non-legume host are highly oxygen-sensitive for their nitrogenase activity, (ii) the Parasponia nodule supernatant increases the oxygen tolerance of the bacteroids.
Cells of the green alga Golenkinia, pre-cultured so that the contribution of any nitrogen source other than delta-aminolevulinic acid (ALA) to endogenous nitrogen reserves is minimal or absent, are capable of sustained growth on ALA as the sole source of nitrogen in either light or dark. Yield is proportional to ALA concentration up to 10 mM; higher concentrations are increasingly inhibitory. This optimal concentration supports cell numbers, growth rates and chlorophyll levels equal to those found using the same concentration of nitrate. These findings support the suggestion that ALA is not committed to porphyrin synthesis and indicate that conclusions regarding regulation of chlorophyll synthesis based on long-term addition of ALA to systems sensitive to carbon/nitrogen levels in the medium be reconsidered.
The polypeptide composition of chloroplast envelopes from wild-type Arabidopsis was compared with that of a mutant (CS1562) deficient in chloroplast dicarboxylate transport activity. An abundant 42 kilodalton (kd) protein in the envelopes from wild-type is low or absent in envelopes from the mutant. It is suggested that this polypeptide may be a component of the dicarboxylate transporter.
A study has been made of the RNA and protein synthesising systems of wheat embryos isolated from seed lots having high viability but differing in vigour. The rate of RNA and protein synthesis in wheat embryos during the early hours of germination is related to the vigour of the seed lot. The imposition of a stress factor, in the nature of a sub-optimal germination temperature, during germination of isolated wheat embryos magnifies the differences in rates of protein and RNA synthesis between high and low vigour seed. Using cell-free protein synthesising systems it has been demonstrated that an important difference between high and low vigour embryos lies in the relative levels of messenger RNA in the embryo. High vigour embryos contain relatively higher levels of poly A+-RNA (i.e. potential mRNA species) than lower vigour embryos and furthermore the level of poly A+-RNA in high vigour embryos increases during early germination whilst in lower vigour embryos the level decreases. The difference in poly A+-RNA levels accounts, at least partially, for the differences in rates of protein synthesis observed between embryos from high and low vigour wheat seed during early germination at both optimal and sub-optimal germination temperatures.
A laboratory study was carried out using field grown peach (Prunus persica) and prune (P. domestica) flowers. The object was to find out why prune flowers are more freeze tolerant than peach flowers. After the flowers are fully open, it was found that the ovaries may still supercool, even with ice crystals present in the flower stem. The mechanics involved were explored with a computer model of the simultaneous heat, water and solute flow in the flower and stem tissue during freezing. Water flow toward growing ice crystals may cause a discontinuity in the liquid phase between the flower stem and the ovary, creating a barrier to nucleation. It was concluded that the prune flowers survive lower temperatures than the peach because the water in their ovaries is more apt to supercool, particularly when the dew point of the air is not reached.
A γ gliadin cDNA clone has been sequenced. The clone encodes 251 amino acid residues beginning with a 19 residue signal peptide. The putative processed amino terminus is characteristic of a γ gliadin. Fourteen imperfect repeats of a heptapeptide occur near the amino terminus. Data from hybrid selected translation suggest that the cDNA contains sol34 of the mRNA coding region. These results provide the first description of the amino terminus of a γ gliadin precursor.
Illumination of radish seedlings with far-red light brings about an increase of the polyribosome content of cotyledons and hypocotyls. Poly(A)-containing RNA from cotyledons of both etiolated and far-red light-treated radish seedlings were isolated and translated in a rabbit reticulocyte lysate. The comparison of translation products showed that the relative levels of translatable mRNA coding for six abundant polypeptides were higher in the extracts of irradiated seedlings. The comparison of mRNA populations was also investigated with respect to organ specificity, polyribosomal localization and inhibition with the cap analoge, 7-methylguanosine 5′-phosphate (pm7G).
We hybridized a 887 bp chloroplast DNA fragment from Chlamydomonas carrying part of the large subunit (LSU) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) to DNA extracted from various Euglena strains. The hybridization patterns observed with several restriction enzyme analyses of Euglena chloroplast DNA are in agreement with the known localization of the Euglena LSU gene (Stiegler et al., Nucleic Acids Res., 10 (1982) 3427). We obtained hybrids with DNA from four wild-type strains and six mutants in which we had characterized RuBisCO. No hybrid was observed with DNA from mutants Y3BUD, W3BUL and W34ZUD in which no RuBisCO was detected, suggesting that the LSU gene is either strongly modified or absent in these mutants.
From nine cultivars of Zea mays L. through in vitro culture of segments of seedlings from matured seeds, we tried to regenerate plants. The following factors were taken in consideration: (1) type of seed germination; (2) excision zone; (3) position of the inoculum in the medium.
Dogwood (Cornus sericea L.) leaves and stems rapidly increased in freezing tolerance during 24 h following transfer from 25/20°C day/night (WT) to 2/2°C day/night (LT). Increases in hardiness continued at a slower rate during the next 6 days of LT exposure. The increase in hardiness during LT exposure was not related to fatty acid composition or degree of fatty acid unsaturation. Fatty acid composition of leaves did not change following transfer from WT to LT over a 7-day period. Linoleic acid accumulated in stem cortical tissue during the 7-day LT-exposure. The elevation of linoleic acid content at LT resulted in an increase in the overall degree of unsaturation in the stem cortical tissue.
This paper describes a new method for improving both the stability and reproducibility of chloroplast enzyme light-activation systems. Usually, the most labile components of these systems are the thylakoids, the preparation of which must be repeated daily. Freezing the thylakoids in small aliquots in liquid nitrogen and storing them at −90°C in a buffer containing 50% glycerol results in preparations whichare completely stable over an 18-month-period. Enzyme light-activation rates were essentially identical with either frozen or fresh thylakoids. Freezing, however, resulted in a slow decline of NADP-protoreduction rates and also in a gradual uncoupling of non-cyclic photophosphorylation.
The induction of an efficient [1-14C]oleoyl-phosphatidylcholine (PC) desaturase activity in microsomes from 16-h-aged slices of potato tubers is dependent on the physiological state of the tubers. At the onset of dormancy, just after harvest, no desaturation activity could be induced even by aging the slices. When stored for 3 months at 4°C, the desaturation activity could be induced by a 16-h-aging of the tuber slices after a lag phase of 18 days at 20°C; this lag was required to break completely the dormancy of the tubers. When sprouting began, the lag phase was shorter (11 days) and later on, no lag phase at all was required to obtain high levels of desaturase activity. On the contrary, oleoyl-CoA hydrolysis and the integration of oleate into PC were independent of the physiological state of tubers.
A procedure for the rapid, large scale preparation of leaf mesophyll chloroplasts from Zea mays by mechanical methods is described. Centrifugation through a Percoll density layer is used as a final purification step, yielding chloroplasts showing 80–90% intactness and high rates of substrate-dependent oxygen evolution under illumination. Thus, these chloroplasts are of similar functional integrity to those prepared from C4 leaf mesophyll protoplasts but the method allows a considerable saving in time and greater yields. The method has been found suitable for mesophyll chloroplast preparation from several other C4-species.
Cell lines of Medicago sativa have been selected for resistance to the toxin(s) produced by Fusarium oxysporum f. sp. medicaginis. Selected cell lines showed resistance in vitro on media containing F. oxysporum culture filtrate. Plants regenerated from resistant cell cultures showed resistance to the pathogen in two greenhouse studies. In addition, cell lines reinitiated from regenerated plants showed resistance on the media containing culture filtrate. Initial resistant selections from long-term culture showed elevated ploidy levels (octoploid and hexaploid). Subsequent selections of resistant cell lines from a shorter selection cycle yielded resistant plants with the normal tetraploid chromosome number.
Calli were obtained from protoplasts of plants of three cultivars of Cucumis melo L. grown under axenic conditions. The composition of the culture medium in which the axenic plants were grown greatly influenced the rate of recovery of protoplasts capable of undergoing sustained divisions. Under the best conditions, cell wall regeneration was accomplished by up to 90% of the protoplasts and the first divisions were observed within 3–4 days of culture. After 20 days, the percentage of dividing cells was 26% in the cultivar 'Valenciano Pinyonet', 21% in the cv. 'Pinyonet Piel de Sapao' and 52% in the cv. 'Cantaloup Charentais'. Two subsequent cultures on media with lower molarity led to calli with elevated growth rates. When plants of the first two cultivars were cultivated in a suboptimal medium, preculture of the leaves was essential in order to obtain protoplasts with mitotic activity.
