Kinetics of the appearance of mRNA for light-harvesting chlorophyll a/b protein in polysomes of barley

Polysomes from dark-grown and illuminated barley seedlings were translated in cell-free systems. The translation products reacting with the antibody against the light-harvesting chlorophyll a/b protein (LHCP) were analyzed by polyacrylamide gel electrophoresis. It was found that, in addition to the precursor protein of LHCP, a product was obtained that co-migrated with the mature protein. Furthermore, the results show that the light-induced proly(A)RNA for LHCP is integrated into the polysomal complex without delay, indicating that the integration of LHCP into the membrane is controlled at a higher level of gene expression.     

Chloroplast photooxidation affects the accumulation of cytosolic mRNAs encoding chloroplast proteins in maize

Maize (Zea mays L.) seedlings were grown in the presence or absence of an herbicide, norflurazon (4-chloro-5-(methylamino)-2-(,,-trifluoro-m-tolyl)-pyridazinone), which prevents the accumulation of colored carotenoids. In the absence of carotenoids, plants grown in high light incur extensive photooxidative damage to their plastids, but relatively little damage elsewhere. Growth in very low light minimizes chlorophyll photooxidation and allows chloroplast development to proceed. We have previously reported that mRNA encoding light-harvesting chlorophyll a/b protein (LHCP) fails to accumulate in high-light-grown carotenoid-deficient seedlings, but accumulates normally in carotenoid-deficient seedlings grown in low light. Here we extend these results by examining the levels of translatable mRNAs encoding seven additional nuclear-encoded chloroplast proteins. When norflurazon-treated seedlings were grown in low light for 8 d and then transferred to high light for 24 h, three cytosolic mRNAs (plastocyanin, Rieske Fe–S protein, and the 33-kdalton (kDa) subunit of the photosystem II O₂-evolving complex) decreased to less than 1% the amount found in untreated seedlings. Two other mRNAs (NADP malic enzyme, EC 1.1.1.40, and the 23-kDa subunit of the photosystem II O₂-evolving complex) decreased significantly but not to levels as low as the first three. Levels of translatable mRNA for two other chloroplast proteins (pyruvate orthophosphate dikinase, EC 2.7.9.1, and ferredoxin NADP oxidoreductase, EC 1.18.1.2) were not reduced in nonflurazon-treated seedlings after 24 h in high light, but did not show the normal light-induced increase found in untreated plants. Photooxidative damage in the chloroplast thus affects the accumulation of a number of cytosolic mRNAs encoding proteins destined for the chloroplast.Abbreviations Da dalton - FNR ferredoxin NADP oxidoreductase - LHCP light-harvesting chlorophyll a/b-binding protein - poly(A)RNA polyadenylated RNA - PPDK pyruvate orthophosphate dikinase - PSII photosystem II - SDSPAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SSu small subunit (of ribulose-1,5-bisphosphate carboxylase)     

Quantitative analysis of membrane distortions induced by mismatch of protein and lipid hydrophobic thickness

The phase transitional behaviour of bilayers of the phospholipid l--ditridecanoylphos-phatidylcholine is studied as a function of protein content for the reaction center (RC) and an antenna protein (LHCP) of the bacterial photosynthetic apparatus. As membrane and protein are structurally well characterized the experimental results can be quantitatively compared with those of calculations based upon elastic models within the Landaude Gennes-theory. Agreement between theory and experiment demonstrates that dominant elastic forces result from a mismatch of hydrophobic regions of membrane and protein. The data also indicate that RC are present in a monomeric form and LHCP in a highly aggregated form. In addition, the latter protein responds to changes in the lipid environment.     

Polyadenylated mRNA for the light-harvesting chlorophyll a/b protein

In thylakoid membranes isolated from green plants of parsley, pea, and barley, the light-harvesting chlorophyll a/b protein complex (LHCP, mol. weight: 25,000), is a major constituent. Poly(A)RNA isolated from these species was translated in a wheat germ, cell-free system. The in vitro translation products were treated with antibodies raised against the LHCP. This treatment resulted in the precipitation of a precursor protein (mol. weight: 29,000). Poly(A)RNA was also prepared from a cell culture ofPetroselinum that does not develop chloroplasts upon illumination. This poly(A)RNA is capable of stimulating amino acid incorporation in the in vitro translation system, however, it does not direct the synthesis of LHCP.     

Synergism of thidiazuron and benzyladenine in axillary shoot formation depends on sequence of application in Miscanthus X ogiformis ‘Giganteus’

Shoots of Miscanthus X ogiformis Honda Giganteus transferred from medium with benzyladenine (BA) to thidiazuron (TDZ) formed significantly more axillary shoots than shoots grown continuously on either medium, or transferred from TDZ to BA. Shoot formation on axillary shoots transferred from BA-containing media to media with kinetin or isopentenyladenine (2iP) or transferred from media with TDZ, kinetin or 2iP to media with BA, corresponded to the number of shoots formed in the previous subculture. Shoot formation on shoots transferred from medium containing BA to media containing combinations of BA and/or TDZ increased with increasing concentrations of TDZ in the first subculture, whereas shoot formation in the second and third subculture depended on the BA concentration. When shoots were transferred from media with BA to media with TDZ, the time for shoot formation, as well as shoot size, indicate that the combined effect of BA and TDZ is expressed only during the early phase of the subculture. The results suggest that adenine- and phenylurea-type cytokinins have a common binding site in the plant cell, and a model is proposed.Abbreviations BA 6-benzyladenine - CBP cytokinin-binding protein - 2iP isopentenyladenine - KIN kinetin - MS Murashige & Skoog - TDZ thidiazuron     

Thylakoid-protein phosphorylation during the life cycle of Scenedesmus obliquus in synchronous culture

The phosphorylation of thylakoid proteins, which comprise apoproteins of the light-harvesting chlorophyll a/b-protein complex (LHCP), was investigated in vivo and in vitro during the development of Scenedesmus obliquus in synchronous cultures. The in-vitro and in-vivo protein phosphorylation exhibited a maximum activity in cells with maximum photosynthetic capacity (8th hour) and miximum activity in cells with minimum photosynthetic capacity (16th hour). The major phosphorylated polypeptides in vivo were the 24/25-kDa and 28–30-kDa apoprotein of the LHCP, a protein of about 32 kDa, and some smaller polypeptides within the range 10 to 20 kDa. In vitro, the main phosphoproteins were the 28–30-kDa apoprotein and the protein characterized by an apparent molecular weight of 32 kDa. Pulse-chase experiments in vivo established that the latter had the fastest radioactivity turnover of the thylakoidal phosphoproteins.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSII photosystem II Dedicated to Prof. Erwin Bünning on the occasion of his 80^th birthday     

An investigation into the ATP requirement for phosphorylation of thylakoid proteins and for the ATP-induced decrease in the yield of chlorophyll fluorescence in chloroplasts at different stages of development

The phosphorylation of thylakoid membrane polypeptides has been investigated in chloroplasts prepared from peas that had been grown under intermittent light and then exposed to between 4 and 48 h of continuous light. At 4 h, when the ratio of the total amount of labelling of a 9 kDa-polypeptide relative to light-harvesting chlorophyll protein (LHCP) polypeptides was much greater than 1, the affinity for ATP was found to be the same (S_0.5, approx. 100 μM) for both polypeptides. In contrast, in fully greened chloroplasts, when labelling of LHCP was much greater than that of the 9 kDa-polypeptide, the S_0.5 for ATP was 40 μM for LHCP and 500 μM for the 9 kDa-polypeptide. A correlation was observed during development between the affinity for ATP of the 9 kDa-species and its abundance relative to LHCP. It is suggested that these polypeptides compete for phosphorylation by the same protein kinase. Simultaneous assay of the ATP-induced fluorescence decrease at different ATP concentrations revealed a close correlation with LHCP labelling but not with labelling of the 9 kDa-polypeptide. This correlation held irrespective of which polypeptide was the major phosphoprotein.     

Relationships between benzyladenine uptake, endogenous free IAA levels and peroxidase activities during upright shoot induction of Cymbidium ensifoilum cv. Yuh Hwa rhizomes in vitro

Studies were undertaken in order to improve the micropropagation ofCymbidiun ensifolium 'Yuh Hwa. It is easy toinduce rhizomes in vitro in this cultivar but not upright shoots, which areneeded for eventual production of a flowering plant. Exposure ofCymbidium ensifolium 'Yuh Hwa rhizomeexplants in vitro to greater than 2.5 M BA for 12weeks or longer inhibited both elongation of upright, aerial shoots and rootgrowth. Through transfer experiments, it was determined that the commitment toinduction of upright shoots occurred after 10–14 days of exposure to 2.5M BA. BA supplied at 20 M during the first 14days of culture was sufficient to induce upright shoot formation, but shootelongation did not continue under these conditions. Using radiolabelled BA,adenine was found to be a major metabolite found in the rhizome tissue at day14. BA comprised 37% of the total DPM recovered in rhizomes grown in 20M BA compared to that of 61% of the total DPM recoveredfrom rhizomes grown in 2.5 M BA. The total amount of BA takenup per gram fresh weight was similar for both green and etiolated rhizomesgrownin 2.5 M BA. Rhizomes grown in 20 M BA tookupapproximately five times as much BA as those grown in 2.5 MBA.Free IAA levels were highest in the rhizome tip (12.7 ng/g FW) anddecreased to one fourth the value at 1 cm behind the tip. Free IAAlevels increased initially in rhizornes transferred to medium with or without20M BA (although at different amounts) and then decreased. Whenactivated charcoal was added to the medium as well as BA there was no initialincrease in IAA. When 20 M BA was added to the medium, solubleperoxidase activity increased while IAA concentrations remained at about thesame level over a 7-day period. Peroxidase activity in rhizomes eitherdecreasedor remained constant when activated charcoal was added to the medium containingBA.     

Energy transfer in the light-harvesting complex II of Dunaliella tertiolecta is unusually sensitive to Triton X-100

Triton X-100, a detergent commonly used to solubilize higher plant thylakoid membranes, was found to be deleterious to Dunaliella LHC II. It disrupted the transfer of excitation energy from chlorophyll b to chlorophyll a. Based on analysis of pigments and immunoassays of LHC II apoproteins from sucrose density gradient fractions, Triton X-100 caused aggregation of the complex, but apparently did not remove chlorophyll b from the apoprotein. Following solubilization with Triton X-100 only CPI could be resolved by electrophoresis. In contrast, solubilization of Dunaliella thylakoids with octyl--D-glucopyranoside preserved energy transfer from chlorophyll b to chlorophyll a. This detergent also effectively prevented aggregation on sucrose gradients and preserved CPI oligomers, as well as LHCP1 and LHCP3 on non-denaturing gels. Solubilization with Deriphat gave similar results. We propose that room temperature fluorescence excitation and emission spectroscopy be used in conjunction with other biophysical and biochemical probes to establish the effects of detergents on the integrity of light harvesting chlorophyll protein complexes. Methods used here may be applicable to other chlorophytes which prove refractory to protocols developed for higher plants.Abbreviations LHC II light harvesting chlorophyll protein complex associated with photosystem II - LHCP1 and LHCP3 monomeric and oligomeric forms of LHC II, respectively, observed on non-denaturing gels - LiDS lithium dodecylsulphate - PMSF phenylmethylsulfonyl fluoride     

Regeneration of patchouli (Pogostemon cablin Benth.) plants from leaf and node callus, and evaluation after growth in the field

Pogostemon cablin (Benth.) is commercially important for its aromatic patchouli oil. Plants were regenerated through callus culture from leaf and nodal segments. Highest callusing was obtained from leaf explants in Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.8% bacto agar and 2 mg/L NAA + 0.5 mg/L BA. Shoot formation frequency was maximum (83%) with BA at 1.0 mg/L. Regenerated plantlets were rooted on MS medium with auxins. Maximum (90%) rooting was obtained using 0.5 mg/L NAA. Plantlets were grown for 4 weeks in this medium and then transferred to pots containing sterile sand in a moisture saturated glass chamber under laboratory conditions. The established plants were grown in pots filled with a mixture of sandsoilmanure (211) under natural daylight conditions in the field. The total leaf yield was increased in the tissue culture derived plants. These plants were dwarf and had higher specific leaf weight (leaf thickness) and leaf area compared to control plants.Abbreviations BA N⁶-Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - IAA Indole 3-acetic acid - MS Murashige and Skoog (1962) medium - NAA 1-Naphthaleneacetic acid     

Integration of nuclear-encoded proteins into pea thylakoids with different pigment contents

The in vitro membrane integration of the light-harvesting protein of photosystem II (LHCP), the Rieske FeS protein of the cytochrome (Cyt) blf-complex, and the NADPH:protochlorophyllide oxidoreductase (Pchlide reductase) into pea thylakoids with different pigment composition was studied. Pea plants (Pisum sativum L. cv. Kelvedon Wonder) with different contents of chlorophyll (Chl) and carotenoids were obtained by growing the seedlings in a greenhouse or in weak red light with or without the herbicide Norflurazon, an inhibitor of carotenoid biosynthesis. Chloroplasts from untreated and Norflurazon-treated plants grown in weak red light contained approximately 29 and 14% of Chl compared to chloroplasts from untreated plants grown in the greenhouse. The corresponding carotenoid contents were 66 and 5%. Following an integration reaction using LHCP precursor protein and chloroplast lysate, thylakoids from untreated and Norflurazon-treated plants grown in weak red light contained approximately 30 and 5% of protease-protected LHCP, respectively, compared to thylakoids of untreated plants grown in a greenhouse. In contrast to LHCP, the in vitro assembly of the Pchlide reductase was only sligthly reduced in chloroplast lysates of plants grown in weak red light compared to greenhouse-grown plants. In chloroplast lysates of Norflurazon-treated plants, however, the amount of membrane associated, protease-protected Pchlide reductase was reduced to 32% of the amount in untreated plants grown under the same light conditions. In contrast, the integration of the Rieske FeS protein occurred to almost similar levels irrespective of light conditions and herbicide treatments. Reconstitution assays where stroma from Norflurazon-treated plants was added to thylakoids from untreated plants, showed that the herbicide did not affect any stromal component(s) vital for the insertion reaction. Removal of samples during the integration reaction of LHCP showed that no degradation of the protein occurred during the assay. Neither was the assembled protein degraded up to 24 h after the termination of the assay. This indicates that growing plants in weak red light, with or without Norflurazon treatment, mainly affected the primary step in thylakoid assembly of LHCP, i.e. the insertion reaction into the membrane. The results further indicate that proteins normally bound to pigments also require pigments for membrane recognition or integration.     

Preliminary studies of micropropagation of Hopea odorata,a dipterocarp tree

Plantlet development from in vitro cultures of Hopea odorato Roxb. is described. Embryos excised from seeds and cultured on Gamborg's B5 or modified Murashige and Skoog (MS) medium with benzyladenine (BA, 2.2–22.2 M) produced axillary shoots at cotyledonary and/or stem nodes. Shoot production was greatest in germinated embryos on modified MS medium with 8.9 M BA. Excised axillary shoots formed few buds when cultured on medium with BA and limited root development occurred on Woody Plant Medium with naphthaleneacetic acid. Nodal explants from aseptically grown plantlets sprouted axillary shoots in modified MS medium with BA.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - PVPP polyvinylpolypyrrolidone     

Genetic distance between populations

Summary Three strains of McIntosh apple (Malus domestica Borkh.) with growth habits ranging from the standard parent type to extremely compact (dwarf) were grown in vitro as meristem-tip cultures on Murashige and Skoog medium containing a range of concentrations of benzyladenine (BA). All strains exhibited a similar optima (3 to 6 M BA) for maximum shoot proliferation and culture weight increase. However, tolerance to supra-optimal concentration of this cytokinin was related to growth habit. For example, at 10 M BA shoot production rates as a percent of the maximum rates were 90%, 20% and zero for the extreme compact, moderate compact and standard strains, respectively. Comparisons among field trees and meristem-tip cultures of all three strains revealed similarities in growth and development.Summerland Research Station, Contribution No. 532     

Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas

Techniques for the regeneration of Jatropha curcas L. from various explants have been developed. Regeneration from hypocotyl, petiole and leaf explants was evaluated on a range of concentrations of zeatin, kinetin and N⁶ benzyladenine (BA) either singly or in combination with indole-3-butyric acid (IBA). Higher regeneration from hypocotyl and petiole explants was obtained on BA with IBA than on zeatin- or kinetin-supplemented media. Leaf discs from the third expanding leaf exhibited higher regeneration potential than those from the fourth leaf. Independent of the explant type, direct adventitious shoot bud induction was recorded highest on MS medium with 2.22 M BA and 4.9 M IBA. Although the same BA concentration but with reduced IBA concentration (0.49 M) proved effective in callus mediated regeneration from hypocotyl and leaf explants, the petioles required lower concentrations of the two growth regulators (0.44 M BA and 0.49 M IBA). Regenerated shoots could be rooted on growth regulator-free gelled full-strength MS medium. Following simple hardening procedures, the in vitro-raised plants could be transferred to soil and grown to maturity in the field.Abbreviations BA N⁶ benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Benzyladenine induction of buds and somatic embryogenesis in Picea omorika (Pančić) Purk.

Somatic embryogenesis and adventitious bud formation, initiated from shoot explants of Picea omorika is described. Benzyladenine (BA) as the only growth regulator, added to modified Von Arnold and Eriksson medium, induced formation of both adventitious buds and embryogenic tissue. Optimal BA concentration for bud induction was 4.5 M and further bud development and plantlet formation was achieved on growth regulator-free medium. The embryogenic tissue formation was induced when the explants were first grown on the medium with high BA content (22.5 M) and then transferred to medium without growth regulators. Subsequent proliferation of embryogenic tissue was accomplished by subculturing on medium containing 9 M 2,4-dichlorophenoxyacetic acid and 4.5 M BA, and further embryo development was achieved on medium with 12 M abscisic acid. Embryos cultured on growth regulator-free medium formed roots and rooted plantlets were successfully established in soil in the greenhouse.     

Plant regeneration from cultured leaves of Lavandula latifolia Medicus: Influence of growth regulators and illumination conditions

Leaves were obtained from 4-week-old seedlings of Lavandula latifolia Medicus grown in vitro. Leaf explants were then cultured on MS medium supplemented with different concentrations and combinations of the auxins IAA or NAA with the cytokinin BA and maintained under three illumination conditions, 16h photoperiod, darkness or darkness followed by a photoperiod, to assess morphogenic responses. Irrespective of illumination conditions, bud regeneration was achieved only in media containing BA or BA/auxin combinations, with the best results being obtained in the presence of BA and 0.06 or 0.6 M IAA or NAA. A photoperiod of 16h appeared to yield the best response in terms of bud regeneration percentage. High auxin concentrations (6.0 or 11.0 M) inhibited bud differentiation, especially when explants were cultured in darkness. On the other hand, low auxin levels and photoperiod improved shoot development. Excised shoots were induced to form roots by transfer to hormone-free MS medium with macronutrients at half strength. The obtained plantlets were ultimately grown in the greenhouse.Abbreviations BA benzyladenine - BM basal medium - IAA indoleacetic acid - MS Murashige & skoog - NAA -naphthaleneacetic acid     

Differential changes in the synthesis and steady-state levels of thylakoid proteins during bean leaf senescence

During senescence of primary bean leaves (Phaseolus vulgaris), there are differential changes in the rates at which thylakoid proteins are synthesized. In particular, synthesis of the 32 kD herbicide-binding protein continues throughout senescence, whereas formation of the and subunits of ATPase, the 68 kD photosystem I reaction center polypeptide, cytochrome f, cytochrome b₆ and the structural apoprotein of the lightharvesting chlorophyll protein complex (LHCP) declines. Pulse-chase experiments with intact leaves indicated rapid degradation of the 32 kD protein, which is consistent with its known rapid rate of turnover. This degradation was light-dependent and inhibited by DCMU, and the kinetics of degradation were similar for young and senescent membranes. In Coomassie-stained gels, the 68 kD reaction center polypeptide of photosystem I, the and subunits of ATPase and the LHCP were the dominant proteins for all ages of membranes. Western blot analysis indicated that cytochrome f and cytochrome b₆ are selectively depleted during senescence. The data have been interpreted as indicating that translational disruptions in both the cytoplasmic and chloroplastic compartments may contribute to the decline in photosynthetic electron transport in the senescing leaf.