Development of photosynthetic electron transport in Pinus silvestris

Photosynthetic electron transport activity has been measured in chloroplasts isolated from dark-grown seedlings of Pinus silvestris L. and in chloroplasts isolated from seedlings subjected to illumination for periods of up to 48 h. Activities of photosystem 2, photosystem 1 and photosystem 2 plus 1 have been measured. Chloroplasts isolated from dark-grown seedlings showed significant electron transport activity through both photosystems and through the entire electron transport chain from water to NADP. Illumination of the seedlings for only 5 min markedly promoted photosystem 2 activity. The artificial electron donor, diphenylcarbazide. promoted activity in chloroplasts from dark-grown seedlings and in chloroplasts from seedlings illuminated for up to 30 min. In comparison to photosystem 2 and overall electron transport from water to NADP, photosystem 1 activity increased only slightly during illumination. Measurements of electron transport and fluorescence kinetics have confirmed that photosynthetic electron transport capacity is limited on the water splitting side of photosystem 2 in dark-grown seedlings, whereas the primary and secondary electron acceptors of photosystem 2 are fully synthesized and functioning in darkness. Polyethylene glycol must be used as a protective agent when isolating photoactive chloroplasts from secondary needles of conifers. However, the presence of polyethylene glycol, when isolating chloroplasts from dark-grown pine cotyledons, caused a total inhibition of the activity of photosystem 2. The failure of others to show a substantial electron transport activity in chloroplasts from dark-grown Pinus silvestris might depend on their use of polyethylene glycol in the preparation medium and/or on their use of suboptimal reaction conditions for the electron transport measurements.     

Types of the daily course of transpiration rate in seedlings of forest trees

Types of the daily course of transpiration rate were studied in two-year old seedlings ofPinus silvestris L.,Picea excelsa L.,Larix decidua L.,Tilia cordata Mill., andAlnus glu-tinosa (L.) Gaertn. The curves illustrating the daily development of transpiration rate (Figs. 3 - 8) may be grouped in two types, showing a midday maximum (one-peak curves) and a midday depression (two-peak curves). Unlike herbaceous plants, the seedlings ofPinus silvestris L. were found to show, during the night-time, lower but not negligible values for the rate of transpiration (see Figs. 3 and 8). The following times are considered most suitable in determining the rate of transpiration for both herbs and woody species during the daylight: 09.0, 12.0, 14.0, and 17.0 hours. For the forest species seedlings further times are suitable for the night-time, as follows: 23.0, 01.0, and 06.0 hours. The rate of transpiration in the seedlings of Pinus silvestris attained very low levels at 3 and between 6 and 8 a.m. and between 6 and 8 p.m. (see Figs 3 and 8).     

Seasonal Effects on Chlorophyll-Protein Complexes Isolated from P-inus silvestris

The seasonal changes in the relative distribution of P700 chlorophyll-protein complex a₁ and light harvesting chlorophyll-protein complex a/b were studied in a natural stand of Pinus silvestris. Similar measurements were made after artificial photobleaching of chlorophyll in pine seedlings or in isolated pine chloroplasts. The chlorophyll-protein complexes were solubilized by sodium dodecyl sulphate and separated by polyacrylamide gel electrophoresis. When autumn and winter destruction of chlorophyll occurs, the chlorophyll a antenna associated with P700 in photosystem 1 (P700-CPa₁) is relatively more affected than the light harvesting complex, which lacks a reaction centre. These results are further supported by low-temperature fluorescence emission properties of isolated chloroplasts presented in this work and elsewhere. The destruction of chlorophyll in stressing autumn and winter climates is most probably caused by photosensitized oxidation of chlorophyll.     

In vitro adventitious bud formation on isolated seedlings ofPinus silvestris L.

The formation of adventitious buds under the influence of 6BAP has been observed on various aerial organs of culturedPinus silvestris seedlings. The inductive power of this cytokinin was found to depend on the concentration and the physiological age of the explant.     

Effect of White and Red Light on the Pigment Content and Functional Activity in Pine Chloroplasts

The pigment content and rates of primary photosynthetic reactions were determined in chloroplasts of 14-day-old pine (Pinus silvestris L.) seedlings grown in light and darkness. In addition, the functional activities were investigated in chloroplasts from dark-grown seedlings exposed to white, red ( = 670 nm), and red + far-red ( = 748 nm) light. Dark-grown seedlings were capable of performing the Hill reaction, noncyclic photophosphorylation, and phenazine methosulfate–supported photophosphorylation, although the reaction rates in chloroplasts from dark-grown plants were considerably lower than in preparations from light-grown plants. Light treatment of dark-grown seedlings rapidly activated the photoreduction of ferricyanide and photophosphorylation, while the additional accumulation of green pigments started only after a lag period of two hours. Preirradiation of dark-grown seedlings with red light stimulated the formation of pigments, especially chlorophyll b, as well as the functional activity of chloroplasts. When far-red light was applied after red-light exposure, the processes examined were inhibited. It is concluded that accumulation of the light-harvesting complex and functional activities of chloroplasts at the photosystem II level in pine seedlings are controlled by the phytochrome.     

Frost Damage Resulting from Ice Crystal Formation in Seedlings of Spruce and Pine

The influence of ice crystal formation in tissues of unhardened and hardened seedlings of Pinus silvestris L. and Picea abies (L.) Karst. was determined. An apparatus for this purpose was constructed. Unhardened seedlings of spruce collapsed completely as a result of ice crystal formation in their shoots, while unhardened seedlings of pine survived ice crystal formation with or without injury to the needles. After a hardening period of 4–5 weeks, seedlings of spruce and pine survived ice formation but injuries occurred in spruce. For pine, on the contrary, the temperature after ice formation had a decisive effect on the extent of the injuries. Observed injuries are discussed in terms of inter- and intracellular ice crystal formation.     

The Effects of Simazine, Applied for Weed Control, on the Mycorrhizal Development of Pinus Seedlings

The herbicide Simazine is widely used in forestry nurseries.Its effect on the incidence of ectomycorrhizae on pine seedlingroot systems was investigated. Under field (nursery) conditions,at routine rates of application, Simazine does not inhibit mycorrhizaldevelopment in Pinus sylvestris and P. nigra, and it may, undersome conditions, actually enhance mycorrhizal formation. Sucha stimulatory effect was also obtained with pot-grown seedlingstreated with the herbicide. It is suggested that soil type andclimatic factors might be involved in determining the responseto Simazine. The mode of stimulation of mycorrhizal developmentby this herbicide is not understood, but it is not via suppressionof weed growth. Pinus sylvestris, Pinus nigra, pine seedlings, ectomycorrhizae, Simazine     

Photophosphorylation and Ultrastructural Development in Pinus nigra Chloroplasts, Grown under Different Spectral Composition of Light

Developed pine seedlings synthesize chlorophyll in darkness. Their photosystem II reducing capacity is very low. The development of chloroplast structure and of photophosphorylation ability has been studied in plastids isolated from Pinus nigra (var. austriaca) developed for 14 days under different spectral compositions of light as compared with chloroplasts isolated from seedlings grown under white light or in darkness. Chloroplast structure was studied by electron microscopy. Cyclic and non-cyclic photophosphorylation were studied under white light. The spectral bands which are efficient for the development of granal structures are different from those needed to make photosystem II functional: red light makes photosystem II functional but does not permit the formation of granal structure, and under yellow light the granal structure develops but photosystem II is not fully functional. Orange light alone fulfils both these purposes. The spectral band around 650 nm seems effective in making the photosystem II functional.     

The Formation of Terpenoids and Their Role in the Metabolism of Pinus halepensis Mill

The results of chemical and ultrastructural research show thatin seedlings of Pinus halepensis growing on vermiculite andwater, nourished by the endosperm, the synthesis of resin proceedsas well in darkness as in light. Starved seedlings, which hadused all the endosperm, were grown without nutrients for tenadditional days in darkness. In these seedlings the quantityof terpenes was greater than in seedlings which were still nourishedby the endosperm. In spite of the long period of starvation,which lowered the density of the cytoplasm and of the numberof the organelles (in epithelial and other cells), the plantsdid not use the terpenoid constituents of the resin as substrates. Pinus halapensis Mill., resin synthesis, terpenoids     

Structural and functional relationships in developing Pinus silvestris chloroplasts

The light dependent chloroplast development of dark grown seedlings of Pinus silvestris L. was followed by analyses of chlorophyll content, chlorophyll a/b ratios, chlorophyll/P700 ratios, chlorophyll-protein complexes and structural changes. Low-temperature fluorescence emission spectra of isolated chloroplasts and separation of sodium dodecyl sulphate solubilized chlorophyll-protein complexes by gel electrophoresis showed that the chlorophyll-protein complexes of photosystem 1 (P700-CPa), photosystem II (PS II-CPa) and the light-harvesting complex LH–CPa/b were present in dark grown seedlings. The low-temperature fuoorescence emission maxima of isolated P700–CPa and PS II–CPa shifted towards longer wavelengths during greening in light, indicating a light induced change of the chlorophyll organisation in the two photosystems. Illumination caused LH–CPa/b to increase relative to P700–CPa, whereas the ratio between LH–CPa/b and PS II–CPa remained essentially constant. Analyses of low-temperature fluorescence spectra with or without 0.01 M Mg²⁺ showed that the Mg²⁺ controlled distribution of excitation energy into PS I was activated upon illumination of the seedlings. The photosynthetic unit size, as defined by the chlorophyll/P700 ratio, did not change over a 96 h illumination period, although the chlorophyll content increased about 6–fold during that time. This result and the constant electron transport rate per unit chlorophyll and time during chlorophyll accumulation provided evidence for a sequential development of the photosynthetic units when illuminating dark grown pine cotyledons. Electron micrographs showed that exposure of dark grown seedlings to light for 2 h caused the prolamellar body to disappear and grana to form. These changes occurred prior to substantial accumulation of chlorophyll or change in the ratio between LH–CPa/b and P700–CPa. However, both the water-splitting system of photosystem II and the Mg²⁺ controlled redistribution of excitation energy was activated during this period.     

Chloroplast development in 4-thiouridine-cultured radish seedlings III. Photochemical activities in isolated plastids

Accumulation of chlorophyll, development of photosystem I andII activities and contents of chloroplastic components wereinvestigated in greening radish seedlings germinated and grownwith 4-thiouridine (4SU). The development of photosystem I activityprior to that of photosystem II was observed also in the 4SU-culturedgreening radish cotyledons in which chlorophyll accumulationwas inhibited up to 60–80% of that of the control. Photochemicalactivities expressed on a plastid protein basis decreased withthe increase of 4SU in the culture medium. In contrast to ferredoxinand ferredoxin-NADP reductase, which were present in significantamounts in the treated cotyledons, chloroplastic cytochromes(f, b₅₅₉ and b₆ decreased in the plastids from 4SU-culturedcotyledons. These results suggest that 4SU interferes in partwith protein synthesis in plastids and thereby with chloroplastdevelopment. (Received December 4, 1979; )     

Intracellular Transport and Elimination of Resin from Epithelial Duct-cells of Pinus halepensis

The ultrastructure of the resin secreting cells of root ductsof young Pinus halepensis seedlings was studied. It is suggestedthat the endoplasmic reticulum (ER) in addition to taking partin resin synthesis also plays a role in transporting the resinfrom the plastids, mitochondria and nuclear envelope to theplasmalemma. By fusing with the plasmalemma the ER releasesthe resin to the outside of the protoplast. The resin producedin the ground cytoplasm and by the Golgi apparatus seems tobe eliminated by plasmalemma invaginations. Pinus halepensis, resin secretion, root ducts, endoplasmic reticulum     

Nutrient Supply, Dry-matter Production and Nutrient Uptake of Forest Tree Seedlings

The dry-matter production and nutrient uptake of forest-treeseedlings grown under controlled levels of nutrition in sub-irrigatedsand culture were investigated. Hardwood seedlings attainedgreater dry weight than conifer seedlings, and Pinus radiataa greater dry weight than P. contorta and P. nigra, but thesegreater dry weights were not related to greater leaf/weightratios, although seed size may have been an important influenceon final seedling dry weight. P. radiata was found to have alower requirement for P than P. contorta and P. nigra in oneexperiment. The demand (total nutrient uptake per unit of time) made bythree species for nutrients upon a site was shown to dependmore upon growth-rate per annum than on efficiency (quantityof nutrients required to produce a unit of dry matter). It isconcluded that the ability of tree species to grow satisfactorilyon soils of low nutrient status depends largely on their abilityfor relatively slow growth. On the other hand, species showinggood response to favourable sites may also make satisfactorygrowth on poor sites.     

Effect of Plant Growth Promoting Bacillus Strains on Pine and Spruce Seedling Growth and Mycorrhizal Infection

Rifamycin-resistant derivatives of plant growth promotingBacilluspolymyxa strains L6, Pw-2, and S20 were used to evaluate theinteraction of bacterial–mycorrhizal co-inoculation onpine and spruce seedling growth. We were particularly interestedin determining if the mechanism by which bacteria stimulatedseedling growth depended on the presence of ectomycorrhizae.Mycorrhizal inoculum was introduced by adding 2ml of one ofsix forest floor soil types originating from different spruceand pine stands to seedling containers. Mycorrhizal roots developedin 34% of pine and 27% of spruce seedlings treated with forestsoil, but no differences between forest soils were detected.Most mycorrhizae were formed byWilcoxinasp. (E-strain) (98%for spruce and 67% for pine); small numbers ofAmphinema-like,Myceliumradicis atrovirens, Suillus-like,Thelephora-like, andTuber-likemycorrhizae were also found on pine (27% in total).Thelephora-likefungi comprised 2% of spruce mycorrhize. In the absence of bacterialinoculum, spruce seedling biomass was positively correlatedwith the number of mycorrhizal root tips, but this trend wasnot detected in spruce inoculated with bacteria or in pine.Bacterial inoculation did not influence the mycorrhizal statusof seedlings, but all threeBacillusstrains stimulated growthof both conifer species. Root biomass, in particular, was significantlyenhanced by up to 18% compared with uninoculated controls. Mycorrhizalfungi improved the growth of spruce seedlings, but plant growthpromotion byBacilluswas similar for mycorrizal and non-mycorrhizalseedlings of both species. Our results suggest thatBacillusstrainsL6-16R, Pw-2R, and S20-R enhance conifer seedling growth througha mechanism unrelated to mycorrhizal fungi. Hybrid spruce; Picea glaucaxengelmannii ; lodgepole pine; Pinus contortavar.latifoliaEngelm.; inoculation; Bacillus polymyxa; seedling growth promotion; mycorrhizae     

Development of photosynthetic activities in dark-grown spruce seedlings

The chloroplasts in dark-grown, 5-day-old seedlings of Piceaabies contained doubly stacked primary thylakoid membranes connectedwith prolamellar bodies. The photo-system II reaction centerand photosystem II-associated electron transfer were assembledin the primary thylakoid membranes, but the oxygen-evolvingsystem remained latent unless the seedlings were exposed tolight. The photoactivation of this system was strongly dependenton temperature during pre-illumination. This implies that somethermal process, in addition to the photoprocess, is involvedin the photoactivation of the oxygen-evolving system. ¹Laboratory of Plant Physiology, The Institute of Physical andChemical Research, Wako-shi, Saitama 351, Japan. ²Department of Biology, Fukuoka Dental College, Fukuoka 814,Japan. (Received June 22, 1977; )     

Studies on the Hill reaction of membrane fragments of blue-green algae V. A correlation between the solubilization of a photochemically active chromoprotein (ACP) and the inactivation of photosystem II activity in membrane fragments of Anabaena cylindrica

The relationship between a photochemically active chromoprotein(ACP) (cf. ref. 1) and photosystem II was investigated withmembrane fragments of Anabaena cylindrica, A. variabilis andP. boryaman. ACP was solubilized from membrane fragments of A. cylindricabut not from those of A. variabilis or P. boryanum, when themembrane fragments had been incubated in a dilute buffer andhad lost their Hill or photosystem II activity. In A. cylindrica,ACP-solubilization always occurred, independent of photosystemII inactivation, on incubation of the membrane fragments inmedia without PEG. However, the amount of ACP solubilizationaccompanying photosystem II inactivation was twice that withoutphotosystem II inactivation. The increase in ACP solubilizationaccompanying photosystem II inactivation. The kinetics resembledthose for the decrease in 695 nm fluorescence emitted by membranefragments at — 196?C (cf. 2). The ACP solubilized independent of photosystem II inactivationwas assumed to have been released during disruption of intactcells in the preparation of membrane fragments. The slow ACPsolubilization upon the inactivation of photosystem II was attributedto the pigment being bound to membranes. We assume that thephoto-reactive component of ACP, P690 (cf. 3, 4), is releasedfrom the membranes during photosystem II inactivation, and thatP690 is a component of photosystem II which emits the 695 nmfluorescence at — 196?C. (Received March 22, 1974; )     

Light-independent synthesis of LHC IIb polypeptides and assembly of the major pigmented complexes during the initial stages of Pinus palustris seedling development

Pinus palustris has a greatly reduced need for light to initiate chloroplast development in comparison to angiosperms. Light is not required for chlorophyll synthesis in dark-grown Pinus palustris seedlings. However, embryos do not contain chlorophyll, and synthesis is limited to seedlings having cotyledon lengths between about 0.5 cm and 2.0 cm. The final amount of chlorophyll accumulated by dark-grown seedlings is about one fifth of that in light-grown seedlingsat the same stage. The major light-harvesting chlorophyll a/b-polypeptides of Photosystem II (LHC IIb) are absent in the embryos but begin to accumulate in seedlings of 0.5 cm cotyledon length, irrespective of the light conditions. Although dark-grown seedlings accumulate most of the pigmented complexes seen in light-grown seedlings, there are differences in the subunit structure of some of them. These findings suggest that the majority of the components of the photosynthetic membrane do not require light for induction of synthesis or assembly into complexes, but that the final forms seen in light-grown seedlings may require light.Abbreviations ALA 5-amino levulinic acid - glucoside -D-glucopyranoside - LHC light-harvesting complex - lhc genes encoding LHCs - PS photosystem     

Electron Microscopic Observations of the Structure of Sieve-connexions in the Phloem of Angiosperms and Gymnosperms

The sieve elements of Pinus silvestris L., Sorbus aucupariaL., Vitis vinifera L., and Cucurbita pepo L. have been examinedelectron microscopically in ultra-thin section, and the structuresof the corresponding sieve areas or sieve plates have been describedand compared. In Pinus the sieve areas contain groups of connectingstrands which enter the wall from the lumen side as individualsbut coalesce within it in the median cavity. This cavity hasdeveloped by wall breakdown in the middle lamella and primarywall region and corresponds to the median nodule visible undera light microscope. Neither in this nor in the other speciesobserved is there any visible closing membrane. Structural differences between the four species are shown tosuggest that the major evolutionary trend in the evolution ofspecialized conducting strands has been the enlargement of theconnecting strands from groups of small separate strands toa smaller number of larger strands as the median cavity becomesenlarged to form a canal through the wall. The connecting strands appear invariably to be dense, highlyosmiophilic and continuous with the cytoplasmic surface of thecell. No signs of micropores or of other tubular structure inthe strands have been found. The structures thus revealed aremore nearly compatible with active transport of materials acrossthe sieve plate than they are with any purely physical mechanism.It is suggested that they are incompatible with any mass flowhypothesis.     

The Spectral and Photochemical Properties of Subchloroplast Membrane Particles from Pinus silvestris

Isolated chloroplasts from Pinus silvestris have been fractionated by a combination of digitonin and Yeda-press treatment. Different subchloroplast particles have been isolated by differential centrifugation. The spectral and photochemical properties of the different fractions have been analysed. Photosystem I was enriched in the light particles (high Chl a/Chl b ratios, low Chl/P700 ratios, high F735/F685 ratios, high photosystem I activity and enrichment of long-wavelength absorbing Chl a). The heavy fractions were enriched in photosystem II (low Chl a/Chl b ratios, high Chl/P700 ratios, low F735/F685 ratios and enrichment of short-wavelength absorbing Chl a). The molar ratio Chl/P700 was about 1.5 times greater for Pinus than for Spinacia, and Pinus had relatively less long-wavelength absorbing Chl a compared with Spinacia.