Early Responses of Sodium-Deficient Amaranthus tricolor L. Plants to Sodium Application

Effects of sodium application on sodium-deficient Amaranthus tricolor L. cv Tricolor seedlings were studied. Thirty-day-old A. tricolor seedlings grown without sodium received either 0.5 millimolar of NaCl or KCl, and the changes in the growth rate, chlorophyll concentration, photosynthetic oxygen evolution, and dark-oxygen consumption, and some enzyme activities were compared. Following the sodium treatment, the sodium concentration in the leaves increased from the initial value of 0.4 millimolar to 2 to 3 millimolar within 24 hours, and also the relative growth rate and O₂ evolution were enhanced within 24 hours. The stimulation of O₂ evolution was greater in the upper leaves than in the lower leaves. Although total chlorophyll concentration did not increase significantly, the increase in the chlorophyll a/b ratio was apparent within 24 hours. There were not significant increases in the C₄ photosynthetic enzyme activities; however, nitrate reductase activity increased by 350% by the sodium treatment within 24 hours, and this increase is considered not to be one of the consequences of the improved photosynthesis. Results suggest that the sodium treatment promoted CO₂ and nitrate assimilation resulting in the growth enhancement, and that sodium can be involved in some other functions than C₄ photosynthesis in A. tricolor plants.     

The requirement for sodium as a micronutrient by species having the c(4) dicarboxylic photosynthetic pathway

Six species having characteristics of plants with the C₄ dicarboxylic photosynthetic pathway, Echinochloa utilis L. Ohwi et Yabuno (Japanese millet), Cynodon dactylon L. (Bermuda grass), Kyllinga brevifolia Rottb., Amaranthus tricolor L. cv. Early splendour, Kochia childsii Hort., and Portulaca grandiflora Hook (rose moss), responded decisively to 0.1 milliequivalent per liter NaCl supplied to their culture solutions initially containing less than 0.08 microequivalent per liter Na. Chlorosis and necrosis occurred in leaves of plants not receiving sodium. Portulaca failed to set flower in the sodium-deficient cultures. Under similar conditions Poa pratensis L. (Kentucky blue grass) having characteristics of the C₃ photosynthetic pathway made normal growth and did not respond to the addition of sodium. It is concluded from these results and previously reported work that sodium is generally essential for species having the C₄ pathway but not for species with the C₃ pathway.     

Sodium Stimulates Growth of Amaranthus tricolor L. Plants through Enhanced Nitrate Assimilation

Effects of Na application on the capacity of NO₃⁻ assimilation were studied in Na-deficient Amaranthus tricolor L. cv Tricolor plants. On day 30 after germination, Na-deficient A. tricolor plants received either 0.5 millimolar NaCl or KCl. The level of nitrate reductase activity doubled within 24 hours by the addition of Na and the enhanced level was maintained thereafter. When the plants were exposed to 2 millimolar ¹⁵NO₃⁻, total ¹⁵N taken up by the plants was greater in the Na-treated plants than in the K-treated plants within 24 hours of the Na treatment. Incorporation of ¹⁵N into the 80% ethanol-insoluble nitrogen fraction of the Na-treated plants in the light period was about 260% of those of the K-treated plants indicating greater capacity of NO₃⁻ assimilation in the Na-treated plants. From these results, it was demonstrated that Na application to the Na-deficient A. tricolor plants promoted NO₃⁻ reduction and its subsequent assimilation into protein, resulting in growth enhancement.     

Evolution of o(2) in brown algal chloroplasts

A method is described for the isolation of photosynthetically active chloroplasts from four species of brown algae: Fucus vesiculosis, Nereocystis luetkeana, Laminaria saccharina, and Macrocystis integrifolia. When compared to lettuce and spinach chloroplasts, the algal chloroplasts all showed lower activities for both photosystems II and I. Chloroplasts from all the plants produced H₂O₂, with photosystem I functioning as the O₂ reductant in the light. In contrast to the green plants, however, brown algal chloroplasts strongly reduced O₂ under conditions where both photosystems II and I remain active. Relative variable fluorescence values were lower both in intact plants and chloroplasts of the brown algae than for either spinach or lettuce. It is suggested that although light harvesting activities appear similar in all the plants, details of electron transport in brown algae may differ from those of green plants.     

Effect of light quality on the organization of photosynthetic electron transport chain of pea seedlings

The activity of NADP and O₂ photoreduction by water is essentially higher in chloroplasts isolated from pea seedlings (Pisum sativum L.) grown under blue light as compared with that from plants grown under red light. In contrast, the photoreduction of NADP and O₂ with photosystem I only is practically the same or even lower in chloroplasts isolated from plants grown under blue light. The addition of plastocyanin does not affect the rate or the extent of NADP photoreduction by water in the chloroplasts isolated from plants grown under blue light, whereas it sharply activates NADP reduction in the chloroplasts isolated from plants grown under red light. The extent of the light-induced oxidation of cytochrome f is appreciably higher in chloroplasts isolated from plants grown under blue light. Cytochrome b₅₅₉ plays the predominant role in the oxidoreductive reactions of these chloroplasts. Furthermore, the fluorescence measurements indicate more effective transfer of excitation energy from chlorophyll to the photosystem II reaction center in chloroplasts isolated from plants grown under blue light.     

Effect of Cd and UV-B radiation on polypeptide composition and photosystem activities ofVigna unguiculata chloroplasts

Rates of whole chain and photosystem 2 activities in chloroplasts isolated fromVigna unguiculata L. seedlings grown under ultraviolet-B (UV-B) enhanced radiation were less affected by 3, 6 and 9 mM CdCl₂ for 60 min at 0 °C in the dark than the rates in chloroplasts from control plants grown under normal irradiation. The results are in agreement with changes in contents of chloroplast 55, 47, 43, 33, 29, 27–25, 23 and 17 kDa polypeptides that were significantly lowered at 3, 6 and 9 mM CdCl₂ only in chloroplasts from control plants. On the other hand, in the simultaneous treatment of chloroplast isolated from control plants the UV-B supported the inhibitory effect of all applied concentrations of CdCl₂. The photosystem 1 activity was only marginally affected in the all experimental variants.     

Sodium Stimulates Regeneration of Phosphoenolpyruvate in Mesophyll Chloroplasts of Amaranthus tricolor

Mesophyll chloroplasts were isolated from leaves of a Na⁺-requiringNAD-malic enzyme type, dicotyledonous C₄ plant, Amaranthus tricolorL. The chloroplasts converted pyruvate to phosphoenolpyruvateunder illumination, and the conversion was stimulated by Na⁺.This observation may explain the requirement for Na⁺ of someC₄ plants. ² Present address: Institute for Life Science Research, NihonNohyaku Co., Ltd., Kawachi-Nagano, Osaka, 586 Japan     

Involvement of Singlet Oxygen in 5-Aminolevulinic Acid-Induced Photodynamic Damage of Cucumber (Cucumis sativus L.) Chloroplasts

Cucumber (Cucumis sativus L., cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid and then incubated in the dark for 14 hours. The intact chloroplasts were isolated from the above plants in the dark and were exposed to weak light (250 micromoles per square meter per second). Within 30 minutes, photosystem II activity was reduced by 50%. The singlet oxygen (¹O₂) scavengers, histidine and sodium azide (NaN₃) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of ¹O₂ monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the ¹O₂ quencher, NaN₃. Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN₃ and histidine. Protochlorophyllide was the most abundant pigment accumulated in intact chloroplasts isolated from 5-aminolevulinic acid-treated plants and was probably acting as type II photosensitizer.     

Photosynthetic Reactions in the Marine Alga Codium vermilara: I. CO(2) Fixation and Hill Reaction in Isolated Chloroplasts

Chloroplasts were isolated from the marine alga Codium vermilara (Siphonales). The isolated chloroplasts were active in CO₂ fixation in the light at a rate comparable to the rates obtained by fragments of thalli. Maximal rates of CO₂ fixation by isolated chloroplasts from Codium were obtained in the presence of salt or sorbitol isoosmotic with sea water. The conditions of isolation of Codium chloroplasts are much less stringent than those required for active chloroplasts from higher plants. The isolated chloroplasts comprise a homogeneous population of the intact “class I” type, as based on microscopic observations and on their inability to reduce ferricyanide unless osmotically shocked. The intact chloroplasts are able to reduce p-benzoquinone at a high rate.     

P(700) activity and chlorophyll content of plants with different photosynthetic carbon dioxide fixation cycles

Representative plants containing either the reductive pentose phosphate cycle or the C₄ dicarboxylic acid cycle of photosynthetic carbon dioxide fixation have distinctly different contents of P₇₀₀ and chlorophylls a and b. With leaf extracts and isolated chloroplasts from C₄ cycle plants, the mean value of the relative ratio of P₇₀₀ to total chlorophyll was 1.83 and the mean value of the ratio of chlorophyll a to b was 3.89. The respective values in similar extracts and chloroplasts from pentose cycle plants are 1.2 and 2.78.     

Chloroplastic and mitochondrial GPX genes play a critical role in rice development

Plant glutathione peroxidases (GPX) catalyze the reduction of H₂O₂ or organic hydroperoxides to water, mitigating the toxicity of these compounds to cells. In rice plants, the GPX gene family is composed of five members that are distributed in a range of sub-cellular compartments including cytosol, mitochondria, chloroplasts, or endoplasmic reticulum. Of these, OsGPX1 and OsGPX4 are located in mitochondria and chloroplasts, respectively. To understand the role of these GPX in rice, the effect of knockdown of OsGPX1 and OsGPX4 in rice plants was evaluated. Our data show that OsGPX4 was essential for in vitro rice regeneration because no plants were obtained from calli carrying a hairpin construct against OsGPX4. Although the knockdown of OsGPX1 did not impair plant regeneration, the plants with silenced OsGPX1 (GPX1s plants) showed reduced shoot length and a reduced number of seeds compared to the non-transformed rice plants. These results indicate that OsGPX1 and OsGPX4 are essential for redox homeostasis which leads to normal growth and development of rice.     

Endophytic fungi associated with Macrosolen tricolor and its host Camellia oleifera

Endophytic fungi play an important role in terrestrial ecosystem, while little is known about those in hemi-parasitic plants, a group of special plants which absorb nutrients from its hosts by haustoria. The relationship of the endophytes in the two parts of the bipartite systems (hemiparasites together with their hosts) is also poorly understood. Endophytic fungi of a hemi-parasitic plant Macrosolen tricolor, and its host plant Camellia oleifera were investigated and compared in this study. M. tricolor contained rich and diversified endophytic fungi (H′ = 2.829), which consisted mainly of ascomycetes, distributed in more than ten orders of four classes (Sordariomycetes, Dothideomycetes, Leotiomycetes and Eurotiomycetes) besides Incertae sedis strains (23.2 % of total). In addition, 2.2 % of isolates were identified to be Basidiomycota, all of which belonged to Agaricomycetes. Obvious differences were observed between the endophytic fungal assembles in the leaves and those in the branches of M. tricolor. The endophytic fungi isolated from C. oleifera distributed in nearly same orders of the four classes of Ascomycota and one class (Agaricomycetes) of Basidiomycota as those from M. tricolor with similar proportion. For both M. tricolor and C. oleifera, Valsa sp. was the dominant endophyte species in the leaves, Torula sp. 1 and Fusarium sp. 1 were the dominant endophytic fungi in the branches. The similarity coefficient of the endophyte assembles in the two host was 64.4 %. Canonical correspondence analysis showed that the endophyte assembles of M. tricolor and C. oleifera were significantly different (p < 0.01).     

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C₄ plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C₄ subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O₂ evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O₂ evolution (29 to 58 micromoles O₂ evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.     

Nutrient requirements differ in two Pedicularis species in the absence of a host plant: implication for driving forces in the evolution of host preference of root hemiparasitic plants

Background and Aims

Facultative root hemiparasitic plants generally have a wide host range, but in most cases show an obvious host preference. The reasons for the marked difference in growth performance of hemiparasites when attached to different hosts are not fully understood. In this study, the hypothesis was tested that hemiparasites showing a preference for different hosts have different nutrient requirements.

Methods

Two facultative root hemiparasitic Pedicularis species (P. rex and P. tricolor) with a different host dependency and preference were used to test their responses to inorganic solutes. The effects of nitrogen, phosphorus and potassium on growth of the hemiparasitic plants not attached to a host were determined, using an orthogonal design in pot cultivation under greenhouse conditions. Variables including biomass, shoot nutrient concentration, root:shoot (R:S) ratios and the number of haustoria were measured.

Key Results

As in autotrophic plants, nutrient deficiency reduced dry weight (DW) and nutrient concentrations in the root hemiparasites. Nitrogen and phosphorus significantly influenced growth of both Pedicularis species, while potassium availability influenced only shoot DW of P. rex. Nitrogen had far more effect on growth of P. rex than on P. tricolor, while phosphorus deficiency caused more marked growth depression in P. tricolor than in P. rex. Pedicularis rex grew faster than P. tricolor in a range of nutrient supplies. Different patterns of biomass allocation between the two Pedicularis species were observed. While P. rex invested more into roots (particularly fine rootlets) than P. tricolor, the number of haustoria produced by P. rex was relatively much lower than that produced by P. tricolor, which had a much smaller root system.

Conclusions

The two Pedicularis species differ in nutrient requirements and biomass allocation. Distinct interspecific traits in growth and nutrient requirements can be driving forces for the differential interactions between hemiparasites and their hosts.     

Preparation of chloroplasts from euglena highly active in protein synthesis

Chloroplasts can be obtained by gentle lysis or mild shear of spheroplasts of vitamin B₁₂-deficient Euglena gracilis and then purified by isopycnic sedimentation on gradients of Ludox AM or Percoll. The chloroplasts appear compact and highly refractile by phase contrast or Hoffmann contrast microscopy. Upon incubation with [³H]leucine or [³⁵S]methionine, the chloroplasts incorporate the amino acids into protein at rates that are 100-fold faster than we had previously observed with Euglena and up to 8-fold faster than with chloroplasts of spinach. Euglena chloroplasts prepared by the current procedure are thus qualitatively superior to those previously available from Euglena and at least as active in protein synthesis as chloroplasts from higher plants.     

Artificial targeting of a nucleus-encoded RNA-binding protein AtRZ1a to chloroplasts affects flowering and ABA response of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the fact that many nucleus-encoded RNA-binding proteins (RBPs) are targeted to chloroplasts and play essential roles in RNA metabolism in chloroplasts, the question of whether artificial targeting of a nucleus-encoded RBP to chloroplasts affects chloroplast function and plant growth has never been addressed. In this study, a nuclear zinc finger-containing Arabidopsis RBP, designated AtRZ1a, which was previously shown to play a role in stress response, was artificially targeted to chloroplasts, and the growth and stress response of the transgenic plants were evaluated. Confocal analysis of the cellular localization of the cTP_AtRZ1a protein containing the N-terminal chloroplast transit peptide (cTP) from rubisco small subunit revealed that the cTP_AtRZ1a fusion protein is successfully targeted to chloroplasts. When grown under normal conditions, flowering of the transgenic plants was delayed, and the FLC expression was significantly upregulated in the transgenic plants. Artificial targeting of AtRZ1a to chloroplasts severely inhibited seedling growth of the plants in the presence of ABA by upregulating expression of ABA signaling-related genes ABI3 and ABI4. Taken together, these results suggest that artificial targeting of a nucleus-encoded AtRZ1a to chloroplasts affects the growth and development of Arabidopsis under normal or ABA treated conditions.     

Pb-Induced Avoidance-Like Chloroplast Movements in Fronds of Lemna trisulca L.

Lead ions are particularly dangerous to the photosynthetic apparatus, but little is known about the effects of trace metals, including Pb, on regulation of chloroplast redistribution. In this study a new effect of lead on chloroplast distribution patterns and movements was demonstrated in mesophyll cells of a small-sized aquatic angiosperm Lemna trisulca L. (star duckweed). An analysis of confocal microscopy images of L. trisulca fronds treated with lead (15 μM Pb²⁺, 24 h) in darkness or in weak white light revealed an enhanced accumulation of chloroplasts in the profile position along the anticlinal cell walls, in comparison to untreated plants. The rearrangement of chloroplasts in their response to lead ions in darkness was similar to the avoidance response of chloroplasts in plants treated with strong white light. Transmission electron microscopy X-ray microanalysis showed that intracellular chloroplast arrangement was independent of the location of Pb deposits, suggesting that lead causes redistribution of chloroplasts, which looks like a light-induced avoidance response, but is not a real avoidance response to the metal. Furthermore, a similar redistribution of chloroplasts in L. trisulca cells in darkness was observed also under the influence of exogenously applied hydrogen peroxide (H₂O₂). In addition, we detected an enhanced accumulation of endogenous H₂O₂ after treatment of plants with lead. Interestingly, H₂O₂-specific scavenger catalase partly abolished the Pb-induced chloroplast response. These results suggest that H₂O₂ can be involved in the avoidance-like movement of chloroplasts induced by lead. Analysis of photometric measurements revealed also strong inhibition (but not complete) of blue-light-induced chloroplast movements by lead. This inhibition may result from disturbances in the actin cytoskeleton, as we observed fragmentation and disappearance of actin filaments around chloroplasts. Results of this study show that the mechanisms of the toxic effect of lead on chloroplasts can include disturbances in their movement and distribution pattern.     

Influence of adenosine phosphates and magnesium on photosynthesis in chloroplasts from peas, sedum, and spinach

¹⁴CO₂ photoassimilation in the presence of MgATP, MgADP, and MgAMP was investigated using intact chloroplasts from Sedum praealtum, a Crassulacean acid metabolism plant, and two C₃ plants: spinach and peas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mg²⁺ were present in the assays, their influence upon CO₂ assimilation was also examined. Free Mg²⁺ was inhibitory with all chloroplasts, as were ADP and AMP in chloroplasts from Sedum and peas. With Sedum chloroplasts in the presence of ADP, the time course of assimilation was linear. However, with pea chloroplasts, ADP inhibition became progressively more severe, resulting in a curved time course. ATP stimulated assimilation only in pea chloroplasts. MgATP and MgADP stimulated assimilation in all chloroplasts. ADP inhibition of CO₂ assimilation was maximal at optimum orthophosphate concentrations in Sedum chloroplasts, while MgATP stimulation was maximal at optimum or below optimum concentrations of orthophosphate. MgATP stimulation in peas and Sedum and ADP inhibition in Sedum were not sensitive to the addition of glycerate 3-phosphate (PGA).

PGA-supported O₂ evolution by pea chloroplasts was not inhibited immediately by ADP; the rate of O₂ evolution slowed as time passed, corresponding to the effect of ADP on CO₂ assimilation, and indicating that glycerate 3-phosphate kinase was a site of inhibition. Likewise, upon the addition of AMP, inhibition of PGA-dependent O₂ evolution became more severe with time. This did not mirror CO₂ assimilation, which was inhibited immediately by AMP. In Sedum chloroplasts, PGA-dependent O₂ evolution was not inhibited by ADP and AMP. In chloroplasts from peas and Sedum, the magnitude of MgADP and MgATP stimulation of PGA-dependent O₂ evolution was not much larger than that given by ATP, and it was much smaller than MgATP stimulation of CO₂ assimilation. Analysis of stromal metabolite levels by anion exchange chromatography indicated that ribulose 1,5-bisphosphate carboxylase was inhibited by ADP and stimulated by MgADP in Sedum chloroplasts.

The appearance of label in the medium was measured when [U-¹⁴C] ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, or AMP and their Mg²⁺ complexes. The rate of back exchange was stimulated by the presence of Mg²⁺. This suggests that ATP, ADP, and AMP penetrate the chloroplast slower than their Mg²⁺ complexes. A portion of the CO₂ assimilation and O₂ evolution data could be explained by differential penetration rates, and other proposals were made to explain the remainder of the observations.

     

Synthesis and uptake of cytoplasmically synthesized pyruvate, pi dikinase polypeptide by chloroplasts

Polyadenylated RNA was isolated from maize leaves and translated in vitro. In agreement with a previous report by others, we found among the translation products a 110-kilodalton pyruvate orthophosphate dikinase (PPDK) precursor that is about 16 kilodaltons larger than the polypeptide isolated from cells. This maize PPDK precursor polypeptide was taken up from the translation product mixture by intact spinach chloroplasts and yielded a mature PPDK polypeptide (94 kilodaltons). The uptake and processing support the proposal that the extra 16-kilodalton size of the polypeptide from in vitro translation of maize leaf mRNA represents a transit sequence which is cleaved after its entry into chloroplasts. Moreover, these results provide additional evidence that in vivo in maize leaf cells PPDK polypeptide is synthesized in the cytoplasm and is transported into the chloroplasts.

Location of PPDK in C₃ plant leaves was investigated by immunochemical analysis. Intact chloroplasts were isolated from leaves of spinach, wheat, and maize. A protein blot of stromal protein in each case gave rise to bands corresponding to authentic PPDK polypeptide. This result indicates that PPDK is present in chloroplasts of C₃ plant leaves as it is in the case of C₄ plants.