Comparison of photomixotrophic and heterotrophic callus and suspension cultures of Pinus elliottii. 1. Photosynthetic properties and ultrastructural evidence for coexistence of starch granules and secondary metabolites

Photomixotrophic callus and suspension cultures of salsh pine (Pinus elliottii var. elliottii Engelm.) have been established. Callus tissues contained up to 2.76 g chlorophyll mg^-1 dry wt and suspensions 2.98 g chlorophyll mg^-1 dry wt. Maximum photosynthetic oxygen evolution was 25–32 mol O₂ h^-1 mg^-1 chlorophyll for callus and 35–39 mol O₂h^-1 mg^-1 chlorophyll for suspension, respectively. Photomixotrophic callus was friable with a high moisture content during early and exponential growth, but evolved into a compact and dense tissue during the latter stage of growth. Compact photomixotrophic callus accumulated and deposited secondary metabolites in the central vacuole and developed large starch granules in the chloroplasts. Secondary metabolites were not observed in photomixotrophic suspensions or in heterotrophic calli and suspensions. Photomixotrophic callus contained numerous mitochondria closely associated with well-developed chloroplasts containing 2–6 thylakoids per granum. Heterotrophic callus was characterized by a poorly developed cytoplasm and cup-shaped mitochondria.     

Photosynthesis in enzymatically isolated leaf cells from the CAM plant Sedum telephium L.

A technique has been developed for the enzymatic isolation of leaf cells from the Crassulacean acid-metabolism plant Sedum telephium. The cells exhibited high activity in both ¹⁴CO₂ incorporation (30–70 mol CO₂ mg^-1 chlorophyll h^-1) and O₂ evolution in the presence of bicarbonate (60–110 mol O₂ mg^-1 chlorophyll h^-1). Half-maximum saturation of ¹⁴CO₂ incorporation occurred at a bicarbonate concentration of ca. 2 mM (20 M CO₂) at pH 8.4 and 30°C. Two types of light-dependent O₂ evolution are reported: O₂ evolution in the absence of exogenously supplied bicarbonate (endogenous O₂ evolution), and bicarbonate-stimulated O₂ evolution. Oxygen evolution in the presence of approximately ambient concentrations of CO₂ appeared to be a combination of the endogenous O₂ evolution and O₂ evolution from fixation of the exogenously supplied CO₂.Abbreviations CAM Crassulacean acid metabolism - cirlo chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - RuDP ribulose-1,5-diphosphate     

Initiation and characterization of a cotton (Gossypium hirsutum L.) photoautotrophic cell suspension culture

A heterotrophic cotton (Gossypium hirsutum L. cv. Stoneville 825) cell suspension culture was adapted to grow photoautotrophically. After two years in continuous photoautotrophic culture at 5% CO₂ (balance air), the maximum growth rate of the photoautotrophic cell line was a 400% fresh weight increase in eight days. The Chl concentration was approximately 500 g per g fresh weight.Elevated CO₂ (1%–5%) was required for culture growth, while the ambient air of the culture room (600 to 700 ul CO₂ 1^–1) or darkness were lethal. The cell line had no net photosynthesis at 350 ul 1^–1 CO₂, 2% O₂, and dark respiration ranged from 29 to 44 mol CO₂ mg^–1 Chl h^–1. Photosynthesis was inhibited by O₂. The approximate 1:1 ratio of ribulose 1,5-bisphosphate carboxylase (RuBPcase) to phosphoenolpyruvate carboxylase (PEPcase) (normally about 6:1 in mature leaves of C₃ plants) was due to low RuBPcase activity relative to that of C₃ leaves, not to high PEPcase activity. The PEPcase activity per unit Chl in the cell line was identical to that of spinach leaves, while the RuBPcase activity was only 15% of the spinach leaf RuBPcase activity. RuBPcase activity in the photoautotrophic cells was not limited by a lack of activation in vivo, since the enzyme in a rapidly prepared cell extract was 73% activated. No evidence of enzyme inactivation by secondary compounds in the cells was found as can be found with cotton leaves. Low RuBPcase activity and high respiration rates are most likely important factors in the low photosynthetic efficiency of the cells at ambient CO₂.Abbreviations Chl chlorophyll - COT heterotrophic cotton cell line - COT-P photoautotrophic cotton cell line - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - RuBPcase RuBP carboxylase - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - MX Murashige and Skoog medium with 0.4 mg 1^–1 2,4-D - KT photomixotrophic medium with 1% sucrose - KT^o KT medium with no carbohydrate - KTP^o KT^o medium supplemented with 0.3 M Picloram - CER CO₂ exchange rate - PCER CO₂ exchange rate in the light     

The carboxylase activity of Rubisco and the photosynthetic performance in aquatic plants

Summary Activated carboxylase activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), as well as photosynthetic rates were measured for 42 species of freshwater and marine macrophytes. While the carboxylase activity varied greatly among the species investigated (0.2–12.5 mol CO₂ mg^–1 chlorophyll min^–1), the submersed freshwater plants showed significantly lower activities than emergent, floating leaved or secondary submersed forms. The variability in photosynthetic rates correlated with the carboxylase activity only for the marine macroalgae, and their photosynthesis to carboxylase activity ratios were close to 1. These plants also had a consistently high inorganic carbon transport capability, and it is suggested that ribulose-1,5-bisphosphate carboxylase/oxygenase activity is an important internal factor regulating the photosynthetic capacity within this plant group where, apparently, the internal CO₂ concentration is high and photorespiration is suppressed. Among the freshwater forms, it appears that their much lower inorganic carbon transport ability, rather than their carboxylase activity, limits the photosynthetic process.     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Effect of cytokinin on morphological changes of suspension cultured cells of the moss, Barbula unguiculata

Suspension cultured cells of the moss, Barbula unguiculata, grow actively in both light and dark culture. Light-grown cells contain chlorophyll and exhibit an undifferentiated callus form. When cells are transferred to a dark condition, they develop into protonemata. Protonemata formation in the dark can be inhibited by the addition of 5 M benzyladenine or 6-furfurylaminopurine but is not affected by the addition of 5 M 2,4-dichlorophenoxyacetic acid or naphthalene acetic acid.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - kinetin 6-furfurylaminopurine - NAA naphthalene acetic acid     

Photoautotrophic growth of Marchantia polymorpha L. cells in suspension culture

A cell line of M. polymorpha was grown photoautotrophically in liquid suspension culture using 1% CO₂ in air as sole carbon source. The growth rate in terms of cell dry-weight during the exponential phase was 0.171 and the doubling time was 1.76 d. The rate of increase in chlorophyll was 1.6 times higher than the growth rate. The highest content of chlorophyll was 24 mg g^-1 dry weight, and the photosynthetic activity of the cells in the exponential phase, as calculated from the growth rate, was at least 60 mol mg^-1 chlorophyll h^-1.     

Response of Rhizobium leguminosarum to nickel stress

Rhizobium leguminosarum strain P-5 biovar viciae was sensitive to Ni²⁺ (MIC, 75 M) and showed concentration-dependent Ni²⁺ uptake in a wide concentration range (50–500 M). Ni²⁺ uptake up to a certain threshold limit also increased thiol content (66 nmol mg^–1 protein), proline content (10.85 nmol mg^–1 protein) and urease specific activity (500 nmol min^–1 mg^–1 protein) maximum corresponding to 100 M Ni²⁺ as the external concentration or 151 nmol Ni²⁺ mg^–1 protein as the intracellular buildup. Proline synthesis was stimulated most even at much lower Ni²⁺ concentration (25 M). Higher intracellular Ni²⁺ load neither favoured thiol nor proline biosynthesis nor urease activity. Ni²⁺ requirement of urease was ascertained by using EDTA-grown cells and the addition of bicarbonate (NaHCO₃, 100 mM) to the crude extract. The induction of thiol or proline by Ni²⁺, therefore, reflects the possible strategies adopted by bacterial cells to overcome the environmental stress.     

Changes in Photosystem II fluorescence in Chlamydomonas reinhardtii exposed to increasing levels of irradiance in relationship to the photosynthetic response to light

The effects of a 60 min exposure to photosynthetic photon flux densities ranging from 300 to 2200 mol m^–2s^–1 on the photosynthetic light response curve and on PS II heterogeneity as reflected in chlorophyll a fluorescence were investigated using the unicellular green alga Chlamydomonas reinhardtii. It was established that exposure to high light acts at three different regulatory or inhibitory levels; 1) regulation occurs from 300 to 780 mol m^–2s^–1 where total amount of PS II centers and the shape of the light response curve is not significantly changed, 2) a first photoinhibitory range above 780 up to 1600 mol m^–2s^–1 where a progressive inhibition of the quantum yield and the rate of bending (convexity) of the light response curve can be related to the loss of Q_B-reducing centers and 3) a second photoinhibitory range above 1600 mol m^–2s^–1 where the rate of light saturated photosynthesis also decreases and convexity reaches zero. This was related to a particularly large decrease in PS II centers and a large increase in spill-over in energy to PS I.Abbreviations Chl chlorophyll - DCMU 3,(3,4-dichlorophenyl)-1,1-dimethylurea - F_M maximal fluorescence yield - F_pl intermediate fluorescence yield plateau level - F₀ non-variable fluorescence yield - F_v total variable fluorescence yield (F_M-F₀) - initial slope to the light response curve, used as an estimate of initial quantum yield - convexity (rate of bending) of the light response curve of photosynthesis - LHC light-harvesting complex - P_max maximum rate of photosynthesis - PQ plastoquinone - Q photosynthetically active photon flux density (400–700 nm, mol m^–2s^–1) - PS photosystem - Q_A and Q_B primary and secondary quinone electron acceptor of PS II     

Factors affecting the growth form of Aspergillus terreus NRRL 1960 in relation to itaconic acid fermentation

The effect of medium composition on the growth form of Aspergillus terreus NRRL 1960 in relation to itaconic acid fermentation has been studied. Four types of mycelial pellets were obtained under the conditions used and may be classified as (a) frayed and loose with 0.1–0.5 mm diameter (b) compact with 0.1–0.5 mm diameter (c) loose with 0.5–2.0 mm diameter and (d) compact with 0.5–2.0 mm diameter. Their respective maximum specific rates of formation and yields of itaconic acid, based on 100 g sucrose supplied, were (a) 1.25 mol mg^–1h^–1 and 55–59 g, (b) 0.27–0.43 mol mg^–1 h^–1 and 26–38 g, (c) 0.75–0.90 mol mg^–1 h^–1 and 45–51 g and (d) 0.12 mol mg^–1 h^–1 and 10 g. The presence of Ca²⁺, Zn²⁺ and Fe²⁺ in the basal medium at concentrations of 23.3 mg/100 ml, 0.01 mg/100 ml and 0.006 mg/100 ml respectively were found to be adequate and crucial in obtaining the desired outgrowth for both high production rates and consistent yields of itaconic acid. The further addition of either commercial plaster of Paris or analytical-reagent-grade CaSO₄, especially when activated by heating to 530°C and present in excess of solubility, results in small and frayed pellets, which lead to itaconic acid yields of 55–59 g acid/100 g sugar supplied.     

Measurement of guard-cell respiration rates using Cartesian-diver technique

Dark respiration rates of guard-cell protoplasts of Commelina communis L. were measured over a temperature range (15–30° C) using a Cartesian-diver microrespirometry technique. Measurements were made using a few microliters of suspension medium containing between 400 and 3 700 protoplasts. Respiration rates were approximately linear for at least 1 h at all temperatures. Respiration rates increased rapidly between 20 and 25° C to relatively high levels (6.11·10^-6 mol O₂ h^-1 protoplast^-1=1259 mol O₂ mg^-1 chlorophyll h^-1=22.97 mol O₂ mg^-1 protein h^-1) with no further increases above this temperature. Respiration rates were much lower in protoplasts 15–16 h old than in freshly prepared ones indicating considerable deterioration of their viability over this time period.     

Photosynthetic Response to Irradiance in Valeriana jatamansi Jones,a Threatened Understorey Medicinal Herb of Western Himalaya

Net photosynthetic rate (P _N) of Valeriana jatamansi plants, grown under nylon net shade or under different tree canopies, was saturated with photons at 1 000 mol m^–2 s^–1 photosynthetic photon-flux-density (PPFD), whereas open-grown plants were able to photosynthesise even at higher PPFD, e.g. of 2 000 mol m^–2 s^–1. Plants grown under net shade had higher total chlorophyll (Chl) content per unit area of leaf surface. However, Chl a/b ratio was maximal in open-grown plants, but remained unchanged in plants grown in nylon net shade and under different tree canopies. Sun-grown plants had thicker leaves (higher leaf mass per leaf area unit), higher wax content, and higher P _N than shade grown plants. Thus V. jatamansi is able to acclimate to high PPFD and therefore this Himalayan species may be cultivated in open habitat to meet the ever-increasing industrial demand.     

Effects of CO<Subscript>2</Subscript> concentration on acclimatization and physiological responses of two cultivars of carob tree

This study reports survival and physiological responses of micropropagated Ceratonia siliqua L. cvs. Galhosa and Mulata plants during ex vitro acclimatization under ambient (AC; 330 mol mol^–1) or elevated (EC; 810 mol mol^–1) CO₂ concentration and a photosynthetic photon flux density of 125 mol m^–2 s^–1. CO₂ enrichment during acclimatization did not improve survival rate that was around 80 % for both treatments. Eight weeks after ex vitro transplantation, photosynthetic capacity and apparent quantum yield in acclimatized leaves were higher in comparison with those in in vitro-grown leaves, without any significant difference between CO₂ treatments. Chlorophyll content increased after acclimatization. However, EC led to a decrease in the total amount of chlorophyll in new leaves of both cultivars, compared to those grown at AC. Soluble sugars and starch contents were not markedly affected by growth EC, although starch had significantly increased after transfer to ex vitro conditions. EC induced an increase in the stem elongation and in the effective life of leaves, and a decrease in the number of new leaves.     

Nutrient content and photosynthesis of young yellowing Norway spruce trees (Picea abies L. Karst.) following calcium and magnesium fertilisation

Severely yellowed ten-year-old spruce trees growing in the Vosges Mountains on an acidic soil were fertilised with Magnesium lime during the spring of 1990. The effects of this treatment were assessed 18 months later. A very significant improvement of the mineral status of the trees was detected, with increasing Mg contents in the needles, and as a consequence, reduced yellowing and improved chlorophyll content. Only slight differences with control trees were observed for height increase. Effects of this improved nutrition on photosynthesis were tested measuring net CO₂ assimilation rates and chlorophyll a fluorescence. Light-saturated net assimilation rates of current-year needles were high, reaching 5.3 mol m^–2 s^–1 on a total needle area basis. The improvement in chlorophyll and Mg content had no significant effect on net assimilation rates or on any parameter describing photochemical functions of both current-and previous-year needles. Despite the strong inter-individual variability in needle chlorophyll and Mg contents (ranging from 0.2 to 0.8 mg g^–1 fresh weight, and 0.05 to 0.5 mg g^-1 dry weight respectively), photochemical efficiency of PS II under limiting irradiance only decreased significantly on older needles displaying Mg contents below 0.1 mg g^–1. It is concluded from these results that spruce trees exhibit a high degree of plasticity with regard to Mg deficiency on acidic soils, and that improved Mg nutrition and increased chlorophyll content do not necessarily improve photosynthesis and height growth.Abbreviations A light-saturated net CO₂ assimilation rate (mol m^–2 s^–1) - g_w light-saturated needle conductance to water vapour (mmol m^–2 s^–1) - _wp and _wm pre-dawn and mid-day needle water potential (MPa) - osmotic potential of sap expressed from needles (MPa) - PFD photosynthetic photon flux density (mol m^–2 s^–1) - F_v/F_m photochemical efficiency of PS II after 20 min dark adaptation - F/F_m ^' photochemical efficiency of PS II reaction centres after 10 min at a PFD of 220 mol m^–2 s^–1     

Light quality affects photosynthesis and leaf anatomy of birch plantlets in vitro

Cultures in vitro of Betula pendula Roth were subjected to light of different spectral qualities. Photosynthetic capacity was highest when the plantlets were exposed to blue light (max recorded photosynthesis, 82 mol CO₂ dm^–2 h^–1) and lowest when irradiated with light high in red and/or far-red wave lengths (max recorded photosynthesis, 40 mol CO₂ dm^–2 h^–1). Highest chlorophyll content (2.2 mg dm^–2 leaf area) was found in cultures irradiated with blue light, which also enhanced the leaf area. Morphometric analysis of light micrographs showed that the epidermal cell areas were largest in plantlets subjected to blue light and smallest in those subjected to red light. Morphometric analysis of electron micrographs of palisade cells, showed that the functional chloroplast area was largest in chloroplasts of leaves subjected to blue light and smallest in those exposed to red light. We suggest that light quality affects photosynthesis both through effects on the composition of the photosynthetic apparatus and on translocation of carbohydrates from chloroplasts.     

Soybean leaf photosynthesis in relation to maturity classification and stage of growth

Leaf photosynthetic rates were measured on field-grown soybeans during the 1980 season. Comparisons were made between different cultivars and isolines representative of maturity groups I–IV. Mature, fully expanded leaves at different nodes on the plant were measured in high light to determine which had the highest potential photosynthetic rates at any one time. Successive leaves during the growing season had maximum rates which increased from about 22 mol CO₂ m^-2 s^-1 on 25 June to a peak of 30–44 mol CO₂ m^-2 s^-1 in early August.The persistency and eventual decline in the maximum rate was associated with the maturity group and related dates of flowering, pod fill and onset of senescence. Early maturing cultivars (groups I and II) had higher peak rates (38–44 mol CO₂ m^-2 s^-1) than later maturing cultivars (30–35 mol CO₂ m^-2 s^-1, groups III and IV). However, the photosynthetic rates of early maturing cultivars declined rapidly after attaining their peak, whereas the leaves of later maturing cultivars maintained their photosynthetic activity for much longer.     

Growth,nitrogen fixation,and mass culture of isolatedAnabaena azollae

Summary An independent strain ofAnabaena azollae was evaluated for its potential as a biofertilizer in wetland rice fields. Sustained rapid growth (doubling time=10.5 h) and nitrogenase activity (32 nmol C₂H₄ h^–1 g^–1 chl) was recorded. Mass cultivation (up to 300 litres) for the first time with this species was also achieved.     

Acclimation of Arabidopsis thaliana to the light environment: Changes in composition of the photosynthetic apparatus

Acclimation to changes in the light environment was investigated in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Plants grown under four light regimes showed differences in their development, morphology, photosynthetic performance and in the composition of the photosynthetic apparatus. Plants grown under high light showed higher maximum rates of oxygen evolution and lower levels of light-harvesting complexes than their low light-grown counterparts; plants transferred to low light showed rapid changes in maximum photosynthetic rate and chlorophyll-a/b ratio as they became acclimated to the new environment. In contrast, plants grown under lights of differing spectral quality showed significant differences in the ratio of photosystem II to photosystem I. These changes are consistent with a model in which photosynthetic metabolism provides signals which regulate the composition of the thylakoid membrane.Abbreviations Aac1 gene encoding actin - Chl chlorophyll - F far-red-enriched light (R:FR = 0.72) - FR far-red light - H high light (400 mol · m^–2 · s^–1) - L low light (100 ml · m^–2 · s^–1) - LHCII light-harvesting complex of PSII - Lhcb genes encoding the proteins of LHCII - R red light - Rbcs genes encoding the small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - W white light (R:FR = 1.40) This work was supported by Natural Environment Research Council Grant No. GR3/7571A. We would like to thank H. Smith (Botany Department, University of Leicester) and E. Murchie (University of Sheffield) for helpful discussions.     

Relationship Between Photosystem 2 Electron Transport and Photosynthetic CO2 Assimilation Responses to Irradiance in Young Apple Tree Leaves

The responses to irradiance of photosynthetic CO₂ assimilation and photosystem 2 (PS2) electron transport were simultaneously studied by gas exchange and chlorophyll (Chl) fluorescence measurement in two-year-old apple tree leaves (Malus pumila Mill. cv. Tengmu No.1/Malus hupehensis Rehd). Net photosynthetic rate (P _N) was saturated at photosynthetic photon flux density (PPFD) 600-1 100 (mol m^-2 s^-1, while the PS2 non-cyclic electron transport (P-rate) showed a maximum at PPFD 800 mol m^-2 s^-1. With PPFD increasing, either leaf potential photosynthetic CO₂ assimilation activity (F_d/F_s) and PS2 maximal photochemical activity (F_v/F_m) decreased or the ratio of the inactive PS2 reaction centres (RC) [(F_i – F_o)/(F_m – F_o)] and the slow relaxing non-photochemical Chl fluorescence quenching (q_s) increased from PPFD 1 200 mol m^-2 s^-1, but cyclic electron transport around photosystem 1 (RFp), irradiance induced PS2 RC closure [(F_s – F_o)/F_m – F_o)], and the fast and medium relaxing non-photochemical Chl fluorescence quenching (q_f and q_m) increased remarkably from PPFD 900 (mol m^-2 s^-1. Hence leaf photosynthesis of young apple leaves saturated at PPFD 800 mol m^-2 s^-1 and photoinhibition occurred above PPFD 900 mol m^-2 s^-1. During the photoinhibition at different irradiances, young apple tree leaves could dissipate excess photons mainly by energy quenching and state transition mechanisms at PPFD 900-1 100 mol m^-2 s^-1, but photosynthetic apparatus damage was unavoidable from PPFD 1 200 mol m^-2 s^-1. We propose that Chl fluorescence parameter P-rate is superior to the gas exchange parameter P _N and the Chl fluorescence parameter F_v/F_m as a definition of saturation irradiance and photoinhibition of plant leaves.