Enhanced susceptibility of photosynthesis to low-temperature photoinhibition due to interruption of chill-induced increase of S-adenosylmethionine decarboxylase activity in leaves of spinach (Spinacia oleracea L.)

The possible involvement of polyamines in the chilling tolerance of spinach (Spinacia oleracea L.) was investigated focusing on photosynthesis. During chilling at 8/5C (day/night) for 6 d, S-adenosylmethionine decarboxylase (SAMDC) activity increased significantly in leaves in parallel with the increase in putrescine and spermidine (Spd) content in leaves and chloroplasts. Treatment of leaves with methylglyoxal-bis(guanylhydrazone) (MGBG), an SAMDC inhibitor, resulted in the deterioration of plant growth and photosynthesis under chilling conditions, which was reversed by the concomitant treatment with Spd through the roots. Plants treated with MGBG showed lower photochemical efficiency of PSII than either the control or plants treated with MGBG plus Spd during chilling and even after transfer to warm conditions, suggesting an increase of photoinhibition due to low Spd in chloroplasts. Indeed, MGBG-treated plants had much lower activities of thylakoid electron transport and enzymes in carbon metabolism as well as higher degrees of lipid peroxidation of thylakoid membranes compared to the control. These results indicate that the enhanced activity of SAMDC with a consequential rise of Spd in chloroplasts is crucial for the cold acclimation of the photosynthetic apparatus in spinach leaves.     

乐果对菠菜叶片POD、SOD、CAT活性及MDA含量的影响

研究了喷施乐果后不同时间及不同施用量对菠菜(Spinacia oleracea)叶片POD、SOD和CAT活性及MDA含量的影响;同时检测了喷药后不同时间菠菜中乐果的残留量。结果表明,喷施乐果可促使菠菜叶片中POD和CAT活性增强以及MDA含量明显增加,而SOD活性在部分乐果处理中呈下降趋势;随着喷药后时间的延长,乐果在菠菜中的残留量亦随之减少。     

Immunological Properties of Spinach Glutathione Reductase and Inductive Biosynthesis of the Enzyme with Ozone

Glutathione reductase (GR) was purified from spinach leavesto the homogeneous state, based on native- and SDS-PAGE bindings.The GR had a polypeptide of 60 kilodalton and its absorptionspectrum was similar to that of GR from human erythrocytes.Antibody against spinach GR, prepared from rabbit, inhibitedGR activity, while the non-immune serum had no effect on theenzyme activity. Purified enzyme and crude extracts from spinachleaves produced fused precipitin lines with anti-GR on the Ouchterlonydouble diffusion tests. Although crude extracts from tobaccoand petunia leaves reacted with anti-GR, these precipitin linesfused only partially with the line between purified spinachGR and anti-spinach GR. Moss, fern and Chlorella crude extractsand purified yeast GR produced no precipitin lines with anti-spinachGR. The extractable GR activity increased significantly in 0.07ppm O₃-fumigated spinach leaves, whereas they suffered no visibleinjuries. The results from the immunoblotting method confirmedthat the O₃-induced increase in extractable GR activity is dueto an increase in the protein level of GR. (Received December 17, 1987; Accepted March 16, 1988)     

Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA cloning and expression

Members of the Chenopodiaceae, such as sugar beet and spinach, accumulate glycine betaine in response to salinity or drought stress. The last enzyme in the glycine betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). In sugar beet the activity of BADH was found to increase two- to four-fold in both leaves and roots as the NaCl level in the irrigation solution was raised from 0 to 500 mM. This increase in BADH activity was paralleled by an increase in level of translatable BADH mRNA. Several cDNAs encoding BADH were cloned from a gt10 libary representing poly(A)⁺ RNA from salinized leaves of sugar beet plants, by hybridization with a spinach BADH cDNA. Three nearly full-length cDNA clones were confirmed to encode BADH by their nucleotide and deduced amino acid sequence identity to spinach BADH; these clones showed minor nucleotide sequence differences consistent with their being of two different BADH alleles. The clones averaged 1.7 kb and contained an open reading frame predicting a polypeptide of 500 amino acids with 83% identity to spinach BADH. RNA gel blot analysis of total RNA showed that salinization to 500 mM NaCl increased BADH mRNA levels four-fold in leaves and three-fold in the taproot. DNA gel blot analyses indicated the presence of at least two copies of BADH in the haploid sugar beet genome.     

Phenolase of spinach roots

Phenolase activity is not found in germinating spinach embryos, but it appears in the radicles when the vascular tissues have developed, and then increases progressively. Unlike the two phenolases detected earlier in the chloroplasts, the root enzyme is a single protein with higher MW occurring both in 3000 g precipitate and 28 000 g supernatant fractions. The phenolase in 3000 g fraction is not activated by treatment with detergents and trypsin. The enzyme is contained mainly in xylem parenchymatous cells adjacent to primary vessels. It also occurs to a lesser degree in the dermal parts, including epidermis and cortex. Similar tissue-level distribution patterns of this enzyme are also observed in the roots of other angiosperms, especially in Compositae.     

The effect of sucrose on the rate of de novo sucrose biosynthesis in leaf protoplasts from spinach, wheat and barley

Protoplasts from the leaves of wheat, spinach, and barley were found to synthesize [14C]sucrose from 14CO2 at rates comparable with those of the parent tissue. CO2 fixation and sucrose biosynthesis ceased virtually immediately when the light was switched off. The effect of sucrose pretreatment on the rate of de novo sucrose biosynthesis was found to vary with leaf age and with plant species. Protoplasts from young wheat and spinach leaves showed an apparent stimulation of the rate of sucrose biosynthesis after sucrose pretreatment. In protoplasts from mature leaves of spinach, sucrose pretreatment produced inhibition. After sucrose pretreatment protoplasts from mature spinach leaves showed low rates of CO2 fixation, and sucrose biosynthesis compared with controls. Conversely, with protoplasts from mature leaves of wheat and barley, the rate of CO2 fixation was unchanged and there was little or no effect on the rate of sucrose biosynthesis after sucrose pretreatment. Preincubation with sucrose had no effect on the activity of sucrose-phosphate synthetase (EC 2.4.1.14), cytoplasmic fructose-1,6-bisphosphatase (EC 3.1.3.11), or UDPglucose pyrophosphorylase (EC 2.7.7.9) from spinach leaves. It was concluded that there is no direct feedback inhibition of sucrose on the sucrose biosynthetic pathway in leaves of spinach, wheat, and barley. The mechanism of inhibition of sucrose biosynthesis by sucrose in spinach remains to be elucidated.     

Change in Levels of Acyl Carrier Proteins in Greening Plants

The levels of acyl carrier proteins (ACP) in greening spinachcotyledons and greening oat leaves were examined by immunoblottingwith antiserum raised against spinach ACP I. Two isoforms ofACP, ACP I and ACP II, were found in spinach cotyledons, asthey were in the green leaves. The level of ACP II was higherthan that of ACP I in etiolated cotyledons. The level of ACPI increased markedly with greening. In the greened cotyledons,the major isoform was ACP I as was the case in green spinachleaves. In oat leaves, two isoforms were also identified, oatACPI (about 12kDa) and ACP II (about 17kDa), which cross-reactedwith the antiserum against spinach ACP I, but which were differentfrom spinach ACPs I and II. The levels of oat ACPs I and IIwere very low in etiolated leaves. The increase in levels ofboth ACPs corresponded to the change in the activity of fattyacid synthesis during illumination for 24 h. During furtherillumination for 24 h, the level of ACP II increased a littlein parallel with the change in the activity of fatty acid synthesis,whereas the level of ACP I increased somewhat more. The functionof oat ACPs I and II is discussed in connection with the formationof chloroplast. (Received March 27, 1989; Accepted September 18, 1989)     

Effects of frost-hardening and salinity on glutathione and sulfhydryl levels and on glutathione reductase activity in spinach leaves

In frost-hardened spinach leaves ( Spinucea oleracea L. ev. Vroeg Reuzenblad ) an enhanced content of water-soluble non-protein sulfhydryl compounds was observed. The enhancement was due to higher levels of glutathione as well as to other non-protein-bound sulfhydryl compounds. In addition glutathione reductase activity was increased upon hardening. The affinity of the enzyme for oxidized glutathione was slightly lowered during hardening. The significance of glutathione accumulation during frost-hardening is discussed. Exposure of spinach to NaCl-stress did not affect the levels of glutathione and glutathione reductase of the leaves. In addition the kinetic properties of the enzyme remained unaltered by salinity. It is suggested that glutathione and glutathione reductase activity are not involved in adaptation of spinach to saline conditions.     

Isolation, characterization, and structural organization of 10-formyltetrahydrofolate synthetase from spinach leaves

One-carbon metabolism mediated by folate coenzymes plays an essential role in several major cellular processes. In the prokaryotes studied, three folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) generally exist as monofunctional or bifunctional proteins, whereas in eukaryotes the three activities are present on one polypeptide. The structural organization of these enzymes in plants had not previously been examined. We have purified the 10-formyltetrahydrofolate synthetase activity from spinach leaves to homogeneity and raised antibodies to it. The protein was a dimer with a subunit molecular weight of Mr = 67,000. The Km values for the three substrates, (6R)-tetrahydrofolate, ATP, and formate were 0.94, 0.043, and 21.9 mM, respectively. The enzyme required both monovalent and divalent cations for maximum activity. The 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities of spinach coeluted separately from the 10-formyltetrahydrofolate synthetase activity on a Matrex Green-A column. On the same column, the activities of the yeast trifunctional C1-tetrahydrofolate synthase coeluted. In addition, antibodies raised to the purified spinach protein immunoinactivated and immunoprecipitated only the 10-formyltetrahydrofolate synthetase activity in a crude extract of spinach leaves. These results suggest that unlike the trifunctional form of C1-tetrahydrofolate synthase in the other eukaryotes examined, 10-formyltetrahydrofolate synthetase in spinach leaves is monofunctional and 5,10-methyl-enetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase appear to be bifunctional. Although structurally dissimilar to the other eukaryotic trifunctional enzymes, the 35 amino-terminal residues of spinach 10-formyltetrahydrofolate synthetase showed 35% identity with six other tetrahydrofolate synthetases.     

Comparison of the effect of rapidly and gradually developing water-stress on carbohydrate metabolism in spinach leaves

Abstract. The effect of water-stress on photosynthetic carbon metabolism in spinach ( Spinacia oleracea L.) has been studied in experiments in which water-stress was induced rapidly by floating leaf discs on sorbitol solutions or wilting detached leaves, and in experiments in which water-stress was allowed to develop gradually in whole plants as the soil dried out. In both short- and long-term water stress, the rate of photosynthesis in saturating CO₂ did not decrease until leaf water potential decreased below -1.0 MPa. However, at smaller water deficits there was already an inhibition of starch synthesis, while sucrose synthesis remained constant or increased. This change in partitioning was accompanied by an increase in activation of sucrose-phosphate synthase (revealed as an increase in activity assayed in the presence of low hexose-phosphate and inorganic phosphate, while the activity assayed with saturating hexosephosphates remained unaltered). Water-stressed leaves had a two- to three-fold higher sucrose content at the end of the night, and contained less starch than non-stressed leaves. When leaves were held in the dark, sucrose was mobilized initially, while starch was not mobilized until the sucrose had decreased to a low level; in water-stressed leaves, starch mobilization commenced at a two-fold higher sucrose content. It is concluded that water-stressed leaves maintain higher sucrose and lower starch levels than non-stressed leaves. This response is found in rapid and long-term stress, and represents an inherent response to water deficits.     

Purification and assay of rubisco activase from leaves

Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase protein was purified from spinach leaves by ammonium sulfate precipitation and ion exchange fast protein liquid chromatography. This resulted in 48-fold purification with 70% recovery of activity and yielded up to 18 milligrams of rubisco activase protein from 100 grams of leaves. Based on these figures, the protein comprised approximately 2% by weight of soluble protein in spinach (Spinacia oleracea L.) leaves. The preparations were at least 95% pure and were stable when frozen in liquid nitrogen. Addition of ATP during purification and storage was necessary to maintain activity. Assay of rubisco activase was based on its ability to promote activation of rubisco in the presence of ribulose-1,5-bisphosphate. There was an absolute requirement for ATP which could not be replaced by other nucleoside phosphates. The initial rate of increase of rubisco activity and the final rubisco specific activity achieved were both dependent on the concentration of rubisco activase. The initial rate was directly proportional to the rubisco activase concentration and was used as the basis of activity. The rate of activation of rubisco was also dependent on the rubisco concentration, suggesting that the activation process is a second order reaction dependent on the concentrations of both rubisco and rubisco activase. It is suggested that deactivation of rubisco occurs simultaneously with rubisco activase-mediated activation, and that rubisco activation state represents a dynamic equilibrium between these two processes.     

Influences of magnesium deficiency and cerium on antioxidant system of spinach chloroplasts

Magnesium-deficiency conditions applied to spinach cultures caused an oxidative stress status in spinach chloroplast monitored by an increase in reactive oxygen species (ROS) accumulation. The enhancement of lipids peroxide of spinach chloroplast grown in magnesium-deficiency media suggested an oxidative attack that was activated by a reduction of antioxidative defense mechanism measured by analysing the activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase, as well as antioxidants such as carotenoids and glutathione content. As the antioxidative response of chloroplast was reduced in spinach grown in magnesium-deficiency media, it caused a significant reduction of spinach plant weight, old leaves turning chlorosis. However, cerium treatment grown in magnesium-deficiency conditions decreased the malondialdehyde and ROS, and increased activities of the antioxidative defense system, and improved spinach growth. Together, the experimental study implied that cerium could partly substitute for magnesium and increase the oxidative stress-resistance of spinach chloroplast grown in magnesium-deficiency conditions, but the mechanisms need further study.     

Purification, subunit structure and immunological comparison of fructose-bisphosphate aldolases from spinach and corn leaves

The cytosol and chloroplast fructose-bisphosphate aldolases from spinach leaves were separated by ion-exchange chromatography on DEAE-cellulose, and were purified by subsequent affinity chromatography on phosphocellulose to apparent homogeneity as judged from polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two aldolases had specific activities of 7.2 and 7.8 units mg protein-1. Molecular weight determinations by electrophoresis in sodium dodecyl sulfate gels and by sedimentation velocity centrifugation in sucrose gradients showed that the aldolases contained four subunits of Mr 38 000 and 35 000, respectively. Antibodies against the cytosol and chloroplast aldolase from spinach leaves were raised in a guinea pig and in a rabbit, respectively. In the Ouchterlony double-diffusion test, the two aldolases did not cross-react. A small degree of cross-reaction was observed by a test in which immune complexes were adsorbed to a solid-phase support (Staphylococcus aureus Cowan I cells) and nonbound enzyme activity was determined after centrifugation. These results imply major structural differences between the two spinach leaf aldolases. Only one major aldolase could be resolved on DEAE-cellulose from corn leaves. The aldolase was purified and had a specific activity of 6.4 units X mg protein-1. The corn leaf aldolase cross-reacted with the antiserum raised against the chloroplast enzyme from spinach leaves, but not with the other antiserum. Thus, the corn leaf aldolase could be identified as a chloroplast enzyme. Since aldolase activity is mostly restricted to the bundle sheath cells of corn leaf, it was concluded that it is compartmentalized in the chloroplasts of these cells but not in chloroplasts of the mesophyll cells.     

菠菜叶中存在两种谷氨酰胺合成酶同工酶

运用非变性聚丙稀酰胺凝胶电泳结合活性染色的方法,在菠菜(Spinacia oleracea L．)生长发育过程中,观察到叶片中至少存在2种谷氨酰胺合成酶(GS),其中一种GS的活性随发育进程而逐渐升高,而另一种GS的活性逐渐降低。在不同来源的成熟的菠菜叶片中同样观察到2种GS的存在。     

Comparative studies of the light modulation of nitrate reductase and sucrose-phosphate synthase activities in spinach leaves

We recently obtained evidence that the activity of spinach (Spinacia oleracea L.) leaf nitrate reductase (NR) responds rapidly and reversibly to light/dark transitions by a mechanism that is strongly correlated with protein phosphorylation. Phosphorylation of the NR protein appears to increase sensitivity to Mg²⁺ inhibition, without affecting activity in the absence of Mg²⁺. In the present study, we have compared the light/dark modulation of sucrose-phosphate synthase (SPS), also known to be regulated by protein phosphorylation, and NR activities (assayed with and without Mg²⁺) in spinach leaves. There appears to be a physiological role for both enzymes in mature source leaves (production of sucrose and amino acids for export), whereas NR is also present and activated by light in immature sink leaves. In mature leaves, there are significant diurnal changes in SPS and NR activities (assayed under selective conditions where phosphorylation status affects enzyme activity) during a normal day/night cycle. With both enzymes, activities are highest in the morning and decline as the photoperiod progresses. For SPS, diurnal changes are largely the result of phosphorylation/dephosphorylation, whereas with NR, the covalent modification is super-imposed on changes in the level of NR protein. Accumulation of end products of photosynthesis in excised illuminated leaves increased maximum NR activity, reduced its sensitivity of Mg²⁺ inhibition, and prevented the decline in activity with time in the light seen with attached leaves. In contrast, SPS was rapidly inactivated in excised leaves. Overall, NR and SPS share many common features of control but are not identical in terms of regulation in situ.     

Fractionation of Jerusalem artichoke phenolase by immobilized copper affinity chromatography

Phenolase from tubers of Jerusalem artichoke was fractionated by metal chelate affinity chromatography using copper conjugated to iminodiacetic acid Sepharose 6B. Four fractions obtained after chromatography showed various specific activities, with an increase in activity of 160-fold for the first unbound enzymatic fraction, and 18-fold for a fraction eluted with glycine buffer. Electrophoresis of phenolase fractions in gradient polyacrylamide gel resulted in a pattern consisting of three major groups of bands differing in relative mobilities.     

供氮水平对菠菜营养品质和体内抗氧化酶活性的影响

通过水培实验,研究了供氮水平对菠菜营养品质和抗氧化酶活性的影响.结果表明,供氮水平由4mmol·L^-1增加到8mmol·L^-1,菠菜产量显著增加,叶片中的维生素C(Vc)含量随着供氮浓度由4mmol·L^-1提高到8mmol·L^-1,再提高供氮水平,Vc含量则明显下降.叶片硝酸盐含量随着氮浓度的提高而增加.供氮浓度从4mmol·L^-1增加到8mmol·L^-1,叶片可溶态草酸含量略有下降,再提高供氮水平则明显上升,而草酸总量随供氮水平提高,先显著升高然后略有降低.SOD和POD酶的活性随供氮水平由4mmol·L^-1提高到8mmol·L^-1而增加,再提高供氮水平,酶活性显著下降;CAT活性随供氮水平的增加而降低,叶片MDA含量先降低后显著升高,而游离脯氨酸含量随氮水平的升高而增加.可见供氮水平为8mmol·L^-1时,菠菜叶片具有较高的生物量、Vc含量和抗氧化酶活性,较低的硝酸盐和草酸含量以及较低的MDA和游离脯氨酸含量,表明供氮浓度8mmol·L^-1有利于提高菠菜的产量、营养品质和抗逆能力,是菠菜生长较适宜的供氮水平.     

Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition

In leaves of spinach plants (Spinacia oleracea L.) performing CO₂ and NO₃⁻ assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.     

Activation of latent phenolase during spinach leaf senescence

The observed increase of phenolase activity and of its rate of activation during spinach leaf senescence is due to reduced binding of latent phenolase to the thylakoid membranes and not to de novo synthesis. The same amount of phenolase which is active in isolated thylakoid membranes from senescent leaves can be found in the membranes of non-senescent leaves after activation of latent enzyme. Tracer experiments give evidence that one multiple form which is responsible for the bulk activity in senescent leaves, is synthesized before, but not after the onset of senescence, indicating that pre-existing latent phenolase is converted to easily activating forms.     

Effect of Ultraviolet- B Irradiation on Lipid Peroxidation in Spinach Leaves

Spinach ( Spinacia oleracea Mill. ) cultivar "Huabo No. 1" was grown in an indoor environment and treated with 13.0 kJ' m-1. d-1 of ultraviolet-B (UV-B 280 to 320 nm) to study the effect of UV-B irradiation on flavonoids and lipid peroxidation in spinach leaves. The results showed that enhanced UV-B irradiation decreased the leaf fresh weight and the content of soluble protein and chlorophyll, and induced large accumulation of UV-absorbing flavonoids in the leaves. UV-B irradiation also promoted the production of superoxide radicals (O2-) and malondialdehyde in spinach leaves. However, the ascorbic acid (ASA) level was decreased under UV-B treatment. It was interesting that high peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT) activities in spinach leaves were induced by UV-B irradiation, the former two being more sensitive. It was suggested that UV-B induced the accumulation of O2- resulting in the lipid peroxidation and in mm inhibiting the growth of spinach. However, the increase of UV-absorbing flavonoids and anti-oxidative enzymes induced by high accumulation of 02- could not reverse the process of UV-B damage.