The influence of high levels of brief or prolonged supplementary far-red illumination during growth on the photosynthetic characteristics, composition and morphology of Pisum sativum chloroplasts

Abstract. Peas were grown in controlled environments (12h white fluorescent light. ∼47 μmol photons m^-2 s 1/12 dark, 25 °C), using (1) 15-min far-red illumination at the end of each photoperiod (brief FR) to simulate the increase in the far-red/red ratio near the end of the day, and (2) high levels of supplementary far-red light (red:far-red ratio=0.04) during the entire photoperiod (long-term FR) to simulate extreme shade conditions under a plant canopy. Brief FR illumination led to marked morphological effects attributable to phytochrome regulation, namely, an increase in internodal length, but a decrease in leaflet area, chloroplast size and chlorophyll content per chloroplast compared with the control. Significantly, brief FR illumination had little or no effect on the amounts of the major chloroplast components (ribulose 1.5-biphosphate carboxylase, adenosine triphosphate synthase, cytochrome b/f complex and Photosystem II) relative to chlorophyll or Photosystem I, and the leaf photosynthetic capacities per unit chlorophyll were similar. In contrast, supplementing high levels of far-red light during the entire photoperiod not only led to the phytochrome effects above, but there was also a marked increase in leaf photosynthetic capacity per unit chlorophyll. due to increased amounts of the major chloroplast components relative to chlorophyll or Photosystem I. We hypothesize that supplementary far-red light, absorbed by Photosystem I, induced an increase in the major chloroplast components by a photosynthetic feedback mechanism. In fully greened leaves, we propose that the two photosystems themselves, rather than phytochrome, may be the predominent sensors of light quantity in triggering modulations of the stoichiometries of chloroplast components, which in turn lead to varying photosynthetic capacities.     

Organellar DNA replication inNicotiana tabacum cultured cells

In the diploid vegetative plant cell, the nuclear DNA is present in two copies, whereas the chloroplast and mitochondria genomes are present in a higher and variable copy number. We have studied the replication of the nuclear, chloroplast and mitochondrial DNA in culturedNicotiana tabacum cells using density and radioactive markers. Essentially all the 10 000 chloroplast genomes in a given cell replicate in one cell cycle as do all the mitochondrial DNA molecules. No measurable level of unreplicated organellar DNA molecules can be detected in these cells.     

A ferredoxin-type iron-sulfur protein gene,frx B,is expressed in the chloroplasts of tobacco and spinach

Previously, a ferredoxin-type iron-sulfur protein, frx B protein, was identified in a high-salt extract of the purified thylakoid membrane of Chlamydomonas reinhardtii, a unicellular green alga. Polyclonal antibody was raised against a synthetic pentadecameric peptide with an amino acid sequence corresponding to the highly conserved region of the putative frx B proteins of 3 land plants [21]. In this report, protein(s) reacting strongly and specifically with this antibody was detected in the equivalent high-salt extract prepared from purified chloroplast of spinach and tobacco. One strong reaction polypeptide band from tobacco chloroplast was purified from SDS-polyacrylamide gel and subjected to endoproteinase lys C digestion. The resulting polypeptides were separated by reversed-phase chromatography. N-terminal sequencing of 3 purified polypeptides revealed that the protein is encoded by the frxB gene identified from DNA sequence analysis.     

Optimization of delivery of foreign DNA into higher-plant chloroplasts

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.     

Transformed plants with elevated levels of chloroplastic SOD are not more resistant to superoxide toxicity

The petunia nuclear gene which encodes the chloroplast isozyme of superoxide dismutase, SOD-1, has been fused with an efficient rbcS promoter fragment and 3 flanking region and introduced into tobacco and tomato cells. Transformed plants carrying this chimeric gene have up to 50-fold the levels of SOD-1 which occur in wild-type plants. However, tobacco plants with 30-to 50-fold the normal SOD-1 activity do not exhibit resistance to the light-activated herbicide paraquat. Similarly, tomato plants with 2-to 4-fold increases in SOD-1 do not exhibit tolerance to photoinhibitory conditions known to increase superoxide levels (high light, low temperatures and low CO₂ concentrations). Our data indicate that increasing the chloroplastic SOD level in a plant cell is not sufficient to reduce the toxicity of superoxide.     

Acclimation of barley to changes in light intensity: photosynthetic electron transport activity and components

Barley seedlings (Hordeum vulgare L. Boone) were grown at 20°C with 16 h/8 h light/dark cycle of either high (H) intensity (500 mole m^-2 s^-1) or low (L) intensity (55 mole m^-2 s^-1) white light. Plants were transferred from high to low (H L) and low to high (L H) light intensity at various times from 4 to 8 d after leaf emergence from the soil. Primary leaves were harvested at the beginning of the photoperiod. Thylakoid membranes were isolated from 3 cm apical segments and assayed for photosynthetic electron transport, Photosystem II (PS II) atrazine-binding sites (Q_B), cytochrome f(Cytf) and the P-700 reaction center of Photosystem I (PS I). Whole chain, PS I and PS II electron transport activities were higher in H than in L controls. Q_B and Cytf were elevated in H plants compared with L plants. The acclimation of H L plants to low light occurred slowly over a period of 7 days and resulted in decreased whole chain and PS II electron transport with variable effects on PS I activity. The decrease in electron transport of H L plants was associated with a decrease in both Q_B and Cytf. In L H plants, acclimation to high light occurred slowly over a period of 7 days with increased whole chain, PS I and PS II activities. The increase in L H electron transport was associated with increased levels of Q_B and Cytf. In contrast to the light intensity effects on Q_B levels, the P-700 content was similar in both control and transferred plants. Therefore, PS II/PS I ratios were dependent on light environment.Abbreviations Asc ascorbate - BQ 2,5-dimethyl-p-benzoquinone - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCIP 2,6-dichlorophenolindophenol - H control plants grown under high light intensity - H L plants transferred from high to low light intensity - L low control plants grown under low light intensity - L H plants transferred from low to high light intensity - MV methyl viologen - P-700 photoreaction center of Photosystem I - Q_B atrazine binding site - TMPD N,N,N,N-tetramethyl-p-phenylenediamine Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC. Paper No. 11990 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA.     

Modification of excitation energy distribution to photosystem I by protein phosphorylation and cation depletion during thylakoid biogenesis in wheat

The effects of protein phosphorylation and cation depletion on the electron transport rate and fluorescence emission characteristics of photosystem I at two stages of chloroplast development in light-grown wheat leaves are examined. The light-harvesting chlorophyll a/b protein complex associated with photosystem I (LHC I) was absent from the thylakoids at the early stage of development, but that associated with photosystem II (LHC II) was present. Protein phosphorylation produced an increase in the light-limited rate of photosystem I electron transport at the early stage of development when chlorophyll b was preferentially excited, indicating that LHC I is not required for transfer of excitation energy from phosphorylated LHC II to the core complex of photosystem I. However, no enhancement of photosystem I fluorescence at 77 K was observed at this stage of development, demonstrating that a strict relationship between excitation energy density in photosystem I pigment matrices and the long-wavelength fluorescence emission from photosystem I at 77 K does not exist. Depletion of Mg²⁺ from the thylakoids produced a stimulation of photosystem I electron transport at both stages of development, but a large enhancement of the photosystem I fluorescence emission was observed only in the thylakoids containing LHC I. It is suggested that the enhancement of PS I electron transport by Mg²⁺-depletion and phosphorylation of LHC II is associated with an enhancement of fluorescence at 77 K from LHC I and not from the core complex of PS I.     

豌豆叶绿体脂氧合酶活性与叶片衰老及膜脂过氧化作用的关系

豌豆叶绿体脂氧合酶(LOX)活性在连体叶片衰老过程中变化不大。ABA处理离体叶片2d叶绿体LOX活性升高,处理时间延长活性下降。抗氧化剂α-生育酚、谷胱甘肽、没食子酸丙酯抑制豌豆叶绿体LOX活性。脂质过氧化产物丙二醛对豌豆叶绿体LOX和大豆纯LOX-1的活性均有抑制作用,大豆LOX-1能促进离体豌豆叶绿体膜脂过氧化作用。因此,豌豆叶绿体LOX可能参与叶片衰老过程中叶绿体膜结构和功能的改变,又受膜脂过氧化产物的制约。     

CORRELATED BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES IN NITROGEN-STARVED EUGLENA GRACILIS1

Growth of Euglena gracilis Z Pringsheim under photoheterotrophic conditions in a nitrogen-deprived medium resulted in progressive loss of chloroplastic material until total bleaching of the cells occurred. Biochemical analysis and ultrastructural observation of the first stages of the starvation process demonstrated an early lag phase (from 0 to 9 h) in which cells increased in size, followed by a period of cell division, apparently supported by the mobilization of some chloroplastic proteins such as the photosynthetic CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. The degradation of the enzyme started after 9 h of starvation and was preceded by a transient concentration of this protein in pyrenoidal structures. Protein nitrogen and photosynthetic pigments as well as number of chloroplasts per cell decreased during proliferation through mere distribution among daughter cells. However, after 24 h, when cell division had almost ceased, there was a slow but steady decline of photosynthetic pigments. This was paralleled by observable ultrastructural changes including progressive loss of chloroplast structure and accumulation of paramylon granules and lipid globules in the cytoplasm. These findings reinforce the role of chloroplastic materials as a nitrogen source during starvation of E. gracilis in a carbon-rich medium. The excess of ribulose-1,5-bisphosphate carboxylase/oxygenase acts as a first reservoir that, once exhausted, is superseded by the generalized disassembly of the photosynthetic structures, if the adverse environment persists more than 24 h.     

Further evidence of a cybridization requirement for plant regeneration from citrus leaf protoplasts following somatic fusion

Summary Somatic hybridization experiments in Citrus that involve the fusion of protoplasts of one parent isolated from either nucellus-derived embryogenic callus or suspension cultures with leaf-derived protoplasts of a second parent, often result in the regeneration of diploid plants that phenotypically resemble the leaf parent. In this study, plants of this type regenerated following somatic fusions of the following three parental combinations were analyzed to determine their genetic origin (nuclear and organelle): (embryogenic parent listed first, leaf parent second) (1) calamondin (C. microcarpa Bunge) + Keen sour orange (C. aurantium L.), (2) Cleopatra mandarin (C. reticulata Blanco) + sour orange, and (3) Valencia sweet orange (C. sinensis (L.) Osbeck) + Femminello lemon (C. limon (L.) Burm. f.). Isozyme analyses of PGI, PGM, GOT, and IDH zymograms of putative cybrid plants, along with RFLP analyses using a nuclear genome-specific probe showed that these plants contained the nucleus of the leaf parent. RFLP analyses using mtDNA-specific probes showed that these plants contained the mitochondrial genome of the embryogenic callus donor, thereby confirming cybridization. RFLP analyses using cpDNA-specific probes revealed that the cybrid plants contained the chloroplast genome of either one or the other parent. These results support previous reports indicating that acquisition of the mitochondria of embryogenic protoplasts by leaf protoplasts is a prerequisite for recovering plants with the leaf parent phenotype via somatic embryogenesis following somatic fusion.Abbreviations cp chloroplast - GOT glutamateoxaloacetate transaminase - IDH isocitrate dehydrogenase - mt mitochondria - PEG polyethylene glycol - PGI phosphoglucose isomerase - PGM phosphoglucomutase - RFLP restriction fragment length polymorphism Florida Agricultural Experiment Station Journal Series No. R-04631.