Regulation of Photosynthetic Induction State in High- and Low-Light-Grown Soybean and Alocasia macrorrhiza (L.) G. Don

Alocasia (Alocasia macrorrhiza [L.] G. Don) and soybean (Glycine max [L.]) were grown under high or low photon flux density (PFD) conditions to achieve a range of photosynthetic capacities and light-adaptation modes. The CO2 assimilation rate and in vivo linear electron transport rate (Jf) were determined over a range of PFDs and under saturating 1-s-duration lightflecks applied at a range of frequencies. At the same mean PFD, the assimilation rate and the Jf were lower under the lightfleck regimes than under constant light. The activation state of two, key enzymes of the photosynthetic carbon reduction cycle pathway, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and fructose-1,6-bisphosphatase, and the photosynthetic induction states (ISs) were also found to be lower under flashing as compared to continuous PFD. Under all conditions, the IS measured 120 s after an increase in PFD to constant and saturating values was highly correlated with the Rubisco activation state and stomatal conductances established in the light regime before the increase. Both the fructose-1,6-bisphosphatase and Rubisco activities established in a particular light regime were highly correlated with the mean Jf in that regime. The relationships between enzyme activation state and Jf and between IS and enzyme activation state were similar in soybean and Alocasia and were not affected either by growth-light regime, and hence photosynthetic capacity, or by flashing versus constant PFD. The common relationship between the linear Jf and the activation state of key enzymes suggests that electron transport may be the determinant of the signal regulating IS, at least to the extent that the IS is controlled by the activation state of key stromal enzymes.     

Concurrent measurements of oxygen- and carbon-dioxide exchange during lightflecks inAlocasia macrorrhiza (L.) G. Don

Oxygen and CO₂ exchange were measured concurrently in leaves of shade-grownAlocasia macrorrhiza (L.) G. Don during lightflecks consisting of short periods of high photon flux density (PFD) superimposed on a low-PFD background illumination. Oxygen exchange was measured with a zirconium-oxide ceramic cell in an atmosphere containing 1 600 bar O₂ and 350 bar CO₂. Following an increase in PFD from 10 to 500 mol photons·m^-2·s^-1, O₂ evolution immediately increased to a maximum rate that was about twice as high as the highest CO₂-exchange rates that were observed. Oxygen evolution then decreased over the next 5–10 s to rates equal to the much more slowly increasing rates of CO₂ uptake. When the PFD was decreased at the end of a lightfleck, O₂ evolution decreased nearly instantaneously to the low-PFD rate while CO₂ fixation continued at an elevated rate for about 20 s. When PFD during the lightfleck was at a level that was limiting for steady-state CO₂ exchange, then the O₂-evolution rate was constant during the lightfleck. This observed pattern of O₂ evolution during lightflecks indicated that the maximum rate of electron transport exceeded the maximum rate of CO₂ fixation in these leaves. In noninduced leaves, rates of O₂ evolution for the first fraction of a second were about as high as rates in fully induced leaves, indicating that O₂ evolution and the electron-transport chain are not directly affected by the leaf's induction state. Severalfold differences between induced and noninduced leaves in O₂ evolution during a lightfleck were seen for lightflecks longer than a few seconds where the rate of O₂ evolution appeared to be limited by the utilization of reducing power in CO₂ fixation.Abbreviation PFD photon flux density (of photosynthetically active radiation)     

Acclimation of photosynthesis to low temperature in Spinacia oleracea L. II. Effects of nitrogen supply

The photosynthetic capacity of leaves of N-sufficent plantsof Spinacia oleracea L. increases following transfer a constanttemperature of 10C for 10 d compared to plants maintained at25C. The effects of nitrogen nutrition on this low temperatureacclimation have been investigated in respect of CO₂ assimilation,the activities and activation states of key enzymes and thepartitioning of recently fixed carbon. N-deficiency greatlyrestricted acclimation of photosynthetic CO₂ assimilation tolow temperature at both ambient and at saturating CO₂ concentrations,indicating a restriction on accilmatory changes in both ribulose1,5-bisphosphatecarboxylase-oxygenase (Rubisco) and the reactions of ribulose1,5-bisphosphateregeneration. Nitrogen limitation led to an increase in thepartitioning of recently-fixed carbon into starch. Total proteinincreased during acclimation in both N-sufficient and N-deficientleaves and was much less affected than were the activities ofenzymes. Increases in the activation state of Rubisco and thestromal fructose-1,6-bisphosphatase occurred in response tolow temperature, but increases in the activities of Rubisco,sucrose-phosphate synthase or the cytosolic fructose1,6-bisphosphatasecould not be sustained in N-deficient plants throughout theperiod of acclimation, although the activities of these enzymesdeclined less precipitately than in non-acclimated N-deficientplants. These data are all consistent with the view that increasesin the activities of key enzymes of carbon assimilation area pre-requisite for acclimation to low temperature and thatthese increases are restricted under N-limitation. Key words: Low temperature, nitrogen, photosynthesis, Rubisco, sucrose-phosphate synthase     

Purification and Characterization of Leu-Proteinase, the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves

The leucine specific serine proteinase present in the soluble fraction of leaves from Spinacia oleracea L. (called Leu-proteinase) has been purified by acetone precipitation and a combination of gel-filtration, ion exchange, and adsorption chromatography. This enzyme shows a molecular weight of 60,000 ± 3,000 daltons, an isoelectric point of 4.8 ± 0.1, and a relative electrophoretic mobility of 0.58 ± 0.03. The Leu-proteinase catalyzed hydrolysis of p-nitroanilides of N-α-substituted(-l-)amino acids as well as of chromogenic macromolecular substrates has been investigated between pH 5 and 10 at 23 ± 0.5°C and I = 0.1 molar. The enzyme activity is characterized by a bell-shaped profile with an optimum pH value around 7.5, reflecting the acid-base equilibrium of groups with pK_a values of 6.8 ± 0.1 and 8.2 ± 0.1 (possibly the histidyl residue present at the active site of the enzyme and the N-terminus group). Among the substrates considered, N-α-benzoyl-l-leucine p-nitroanilide shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 1 × 10⁻⁹ molar. In agreement with the enzyme specificity, only N-α-tosyl-l-leucine chloromethyl ketone, di-isopropyl fluorophosphate and phenylmethylsulfonyl fluoride, among compounds considered specific for serine enzymes, strongly inhibit the Leu-proteinase. Accordingly, the enzyme activity is insensitive to cations, chelating agents, sulfydryl group reagents, and activators.     

Esterolytic Properties of Leucine-Proteinase, the Leucine-Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves : A Steady-State and Pre-Steady-State Study

Steady-state and pre-steady-state kinetics for the hydrolysis of p-nitrophenyl esters of N-α-carbobenzoxy(-l-)amino acids catalyzed by leucine-proteinase were determined between pH 5 and 10 (I = 0.1 molar) at 23 ± 0.5°C. For the substrates considered: (a) the acylation step is rate-limiting in catalysis; (b) the pH profiles of k_cat and k_cat/K_m reflect the ionization of two groups with pK_a values ranging between 6.5 and 6.9, and 8.1 and 8.3 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); and (c) values of K_m are pH independent. Among the substrates examined, N-α-carbobenzoxy-l-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 × 10⁻¹⁰ molar at the optimum pH value (approximately 7.5).     

Role of Ascorbate in Detoxifying Ozone in the Apoplast of Spinach (Spinacia oleracea L.) Leaves

Both reduced and oxidized ascorbate (AA and DHA) are present in the aqueous phase of the extracellular space, the apoplast, of spinach (Spinacia oleracea L.) leaves. Fumigation with 0.3 [mu]L L-1 of ozone resulted in ozone uptake by the leaves close to 0.9 pmol cm-2 of leaf surface area s-1. Apoplastic AA was slowly oxidized by ozone. The initial decrease of apoplastic AA was <0.1 pmol cm-2 s-1. The apoplastic ratio of AA to (AA + DHA) decreased within 6 h of fumigation from 0.9 to 0.1. Initially, the concentration of (AA + DHA) did not change in the apoplast, but when fumigation was continued, DHA increased and AA remained at a very low constant level. After fumigation was discontinued, DHA decreased very slowly in the apoplast, reaching control level after 70 h. The data show that insufficient AA reached the apoplast from the cytosol to detoxify ozone in the apoplast when the ozone flux into the leaves was 0.9 pmol cm-2 s-1. The transport of DHA back into the cytosol was slower than AA transport into the apoplast. No dehydroascorbate reductase activity could be detected in the apoplast of spinach leaves. In contrast to its extracellular redox state, the intracellular redox state of AA did not change appreciably during a 24-h fumigation period. However, intracellular glutathi-one became slowly oxidized. At the beginning of fumigation, 90% of the total glutathione was reduced. Only 10% was reduced after 24-h exposure of the leaves to 0.3 [mu]L L-1 of ozone. Necrotic leaf damage started to become visible when fumigation was extended beyond a 24-h period. A close correlation between the extent of damage, on the one hand, and the AA content and the ascorbate redox state of whole leaves, on the other, was observed after 48 h of fumigation. Only the youngest leaves that contained high ascorbate concentrations did not exhibit necrotic leaf damage after 48 h.     

Photoinhibition of Photosynthesis in Sun and Shade Grown Leaves of Grapevine (Vitis vinifera L.)

The degree of photoinhibition of sun and shade grown leaves of grapevine was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (F_v/F_m) and electron transport measurements. The potential efficiency of photosystem 2 (PS2), F_v/F_m, markedly declined under high irradiance (HI) in shade leaves with less than 10 % of F₀ level. In contrast, F_v/F_m ratio declined with about 20 % increase of F₀ level in sun leaves. In isolated thylakoids, the rate of whole chain and PS2 activity in HI shade and sun leaves was decreased by about 60 and 40 %, respectively. A smaller inhibition of photosystem 1 (PS1) activity was also observed in both leaf types. In the subsequent dark incubation, fast recovery was observed in both leaf types that reached maximum PS2 efficiencies similar to non-photoinhibited control leaves. The artificial exogenous electron donors DPC, NH₂OH, and Mn²⁺ failed to restore the HI-induced loss of PS2 activity in sun leaves, while DPC and NH₂OH were significantly restored in shade leaves. Hence HI in shade leaves inactivates on the donor side of PS2 whereas it does at the acceptor side in sun leaves, respectively. Quantification of the PS2 reaction centre protein D1 and the 33 kDa protein of water splitting complex following HI-treatment of leaves showed pronounced differences between shade and sun leaves. The marked loss of PS2 activity in HI leaves was due to the marked loss of D1 protein of the PS2 reaction centre protein and the 33 kDa protein of the water splitting complex in sun and shade leaves, respectively.     

Response of Spinach Leaves (Spinacia oleracea L.) to Ozone Measured by Gas Exchange,Chlorophyll a Fluorescence,Antioxidant Systems,and Lipid Peroxidation

Spinach (Spinacia oleracea L. cv. Clermont) leaves grown in open-top chambers and exposed to three different concentrations of ozone were measured for gas exchange, chlorophyll a fluorescence, antioxidant systems, and lipid peroxidation at the end of growing season. High O₃ concentration reduced F_v/F_m, indicating that the efficiency in the energy conversion of photosystem 2 (PS2) was altered. The rate of non-cyclic electron transport rate and the capacity to reduce the quinone pool were also affected. The development of non-photochemical quenching was not high enough to decrease the photon excess in the PS2. The limitation of photosynthetic activity was probably correlated with stomata closure and with an increase in intercellular CO₂ concentration. Under oxidative stress, superoxide dismutase (SOD) activity was stimulated in parallel with lipid peroxidation. We did not find any differences in the ascorbate (AsA) pool and ascorbate peroxidase (APX) or glutathione reductase (GR) activities between air qualities. Small, but similar responses were observed in spinach leaves exposed to ambient ozone concentration.     

Isolation and Characterization of a Virus Inhibitor from Spinach (Spinacia oleracea L.)

A virus inhibiting protein (VI) was isolated from spinach (Spinacia oleracea L.). The VI inhibited infections of test plants with plus- and minus-strand RNA viruses. Inoculation of both local lesion and systemic hosts with TMV in the presence of varying amounts of the VI resulted in typical dose response curves for the number of local lesions or the amount of virus respectively. The lowest concentration of VI leading to a significant reduction in the number of local lesions was 0.06 μg/ml. The VI was found to inhibit local lesion formation only when applied within 2–3 h p.i. but still reduced the number of local lesions when applied up to 9 h prior to virus inoculation. The antiviral activity could be attributed to a protein of molecular weight 29,000 dalton with an isoelectric point of 10.3. Its activity was destroyed by heating for 30 min to 70°C. These characteristics resemble those of other virus inhibiting proteins described for members of the order Caryophyllales such as the Phytolacca inhibitor against which a serological relationship was obtained.     

Comparative Characteristics of Growth and Photosynthesis of Sun and Shade Leaves from Normal and Pendulum Walnut (Juglans regia L.) Trees

Pendulum walnut leaves exhibited various adaptive responses related to the regulation of photon interception such as specific downward orientation, greater leaf area, and larger pigment pool. Changes in the regulation of PS2 such as higher thermal dissipation (NPQ) and lower quantum efficiency (Φ_PS2) that protect the photosynthetic apparatus against damages were also found. The growth and photosynthetic features of pendulum walnut leaf are interpreted as adaptations that allow the pendulum walnut tree to compensate the impaired ability by appropriate growth to ensure the energy needs for photosynthesis, respectively for biomass formation. This revised version was published online in August 2006 with corrections to the Cover Date.     

菠菜种子胰蛋白酶抑制剂的分离纯化与部分性质研究

以菠菜种子为材料,经脱脂、酸性溶液抽提、热变性、硫酸铵分部沉淀得到胰蛋白酶抑制剂粗提物。再经离子交换、亲和层析和凝胶过滤,分离得到胰蛋白酶抑制剂SOTI,纯化倍数为57．22。SDS-PAGE测定其分子量约为22kD,等电聚焦测定其等电点为4．02。SOTI具有较高的热稳定性,在100℃处理后仍然具有一定的抑制活性。     

cDNA Cloning and Expression of the Plastidic Copper/Zinc-Superoxide Dismutase from Spinach (Spinacia oleracea L.) Leaves

A cDNA clone for copper/zinc-superoxide dismutase (Cu/Zn-SOD)was isolated from spinach (Spinacia oleracea L.) leaves. Itsnucleotide sequence showed that it codes for a precursor polypeptideof 222 amino acids, including the NH₂-terminal 68-residue extensionwhich corresponds to a plastidic transit peptide. Northern hybridization,using plastidic and cytosolic Cu/Zn-SOD cDNAs as the probes,revealed that these two genes are differentially expressed inthe roots and leaves of spinach. ¹Present address: Department of Biochemistry and Microbiology,Cook College, Rutgers University New Brunswick, NJ 08903-0231,U.S.A.     

Similar Susceptibility to Excess Irradiance in Sun and Shade Acclimated Saplings of Norway Spruce [Picea Abies (L.) Karst.] and Stone Pine (Pinus Cembra L.)

Photosynthetica - We compared the responses of sun and shade acclimated saplings of Picea abies and Pinus cembra to excess photosynthetic photon flux density (PPFD) equivalently exceeding the level...     

Similar Susceptibility to Excess Irradiance in Sun and Shade Acclimated Saplings of Norway Spruce [Picea Abies (L.) Karst.] and Stone Pine (Pinus Cembra L.)

We compared the responses of sun and shade acclimated saplings of Picea abies and Pinus cembra to excess photosynthetic photon flux density (PPFD) equivalently exceeding the level for saturating net photosynthetic rate (P _N). Exposure for 2 h up to 2000 μmol(photon) m⁻² s⁻¹ did not affect radiant energy saturated P _N. Photoinhibition of photosynthesis was indicated by a small (10 %) reduction of the potential efficiency of photosystem 2 as derived from measurements of chlorophyll fluorescence (F_V/F_M). However, the extent of F_V/F_M reduction and half-time for recovery were similar in sun and shade acclimated saplings of both species. Furthermore, the effect on F_V/F_M was not stronger when the plants were exposed to excess PPFD at 5 °C instead of 15 °C. Frost-hardening of plants increased slightly their resistance to excess PPFD. Establishment of these conifer saplings usually acclimated to shade in their natural habitat may hardly be endangered by a sudden increase of PPFD, e.g., by gap formation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ecdysteroids in spinach (Spinacia oleracea L.): biosynthesis, transport and regulation of levels

Many plant species produce phytoecdysteroids (PEs: i.e. analogues of insect steroid hormones). There is increasing evidence that PEs are used as a chemical defence by plants against non-adapted insects and nematodes. PEs are good candidates for the development of an environmentally safe approach to crop protection. Most crop species do not accumulate PEs. However, many arguments support the idea that most, if not all, plant species have the genetic ability to produce PEs, but the biosynthetic pathway is not active. A better understanding of the PE biosynthetic pathway and its regulation is consequently necessary. Spinach is one of the very few crop plants which produce large amounts of PEs, of which 20-hydroxyecdysone is the major component. Labeling experiments with radiolabeled precursor (mevalonic acid), putative ecdysteroid intermediates and 20-hydroxyecdysone itself have allowed investigation of PE biosynthesis and transport during spinach development. Biosynthesis takes place in older leaf sets ("sources"), but not in the young developing ones, which in contrast accumulate (acting as "sinks") the PEs produced by the older leaves. PEs are thus continuously redistributed within the developing plant, as its leaf set number increases. The biosynthetic pathway has been analyzed using excised leaves and various labeled precursors, and a preferential sequence of the last steps has been established. Although they do not produce PEs, apical leaf sets are nevertheless able to perform several putative terminal steps of PE biosynthesis. The regulatory mechanisms of PE synthesis appear to involve a direct negative feedback of 20-hydroxyecdysone (the major PE in spinach) on its own synthesis; thus, a sustained synthesis in older leaves requires that they can export the PE they produce.     

Chemical constituents of Physochlaina physaloides (L.) G. Don (Solanaceae)

The study of the whole plants of Physochlaina physaloides (L.) G. Don. has led to the isolation of eight alkaloids (1–6, 18, and 25), fourteen flavonoids (9, 11–17, and 19–24), two coumarins (7 and 10), one iridoid (8). The structures of these compounds were identified spectroscopically, and nuclear magnetic resonance (NMR) data were compared with previously reported data. This is the first study to describe the isolation and identification of compounds 1–3, 7, and 10–25 in P. physaloides. The chemotaxonomic significance of these compounds in the genus Physochlaina and its closely related genera was discussed.     

长春花组织培养条件的最优化

确定出长春花的愈伤组织诱导条件。优化长春花的组织培养的外植体、消毒方法、激素的种类和配比。叶柄作为诱导培养的外植体,2．5％有效氯的次氯酸钠消毒15min-20min,2,4－D：Kt＝2－3．5ppm:1ppm的激素诱导下11d诱导出生长良好、呈疏松颗粒状的愈伤组织。     

野生植物长春花的营养成分

对野生植物长春花的营养成分进行了分析 ,结果表明 ,长春花中含有多种营养成分 ,其中至少含有 17种氨基酸。为开发利用植物资源提供了科学依据