Endogenous ascorbate restrains apoplastic peroxidase activity during sunflower leaf development

Several apoplastic enzymes have been implicated in the control of elongation growth of plant cells. Among them, peroxidases contribute to both loosening and stiffening of the cell wall. They appear to be regulated by various mechanisms, including the action of extracellular inhibitors. To obtain evidence of the role of the enzyme–inhibitor interaction during leaf development, the intercellular washing fluids from Helianthus annuus leaves of different ages were isolated using standard methods of vacuum infiltration and centrifugation. Peroxidase activities, assessed using tetramethylbenzidine as substrate, increased during leaf development, reaching a maximum value after the leaves were fully expanded. An inhibitor, chemically characterised as ascorbate, co‐localised with the enzyme in the apoplast. Moreover, there was a strong negative correlation between the action of peroxidase and the micromolar concentration of ascorbate in the apoplastic fluid. The results show that in growing leaves, the in planta ascorbate concentration is able to restrain peroxidase enzyme activity. Then, at the time of growth cessation, the loss of extracellular ascorbate relieves the inhibition on this enzyme that contributes to wall fixation.     

Cell Walls of Phaseolus vulgaris Leaves Contain the Azocoll-Digesting Proteinase

The leaves of Phaseolus vulgaris L. cv Greensleeves contain an endopeptidase with a pH optimum of 9.0 and an isoelectric point between 10.0 and 10.5. This endopeptidase is the only abundant Azocoll-digesting proteinase in the leaves. The activity of this enzyme is highest in immature leaves and declines as the leaf matures and senesces. Enzymically isolated protoplasts contain very little of this proteinase. The proteinase can be recovered readily from the extracellular fluid obtained by gentle centrifugation of leaf strips vacuum-infiltrated with a buffered solution. These experiments indicate that the Azocoll digesting proteinase is located in the periplasmic space and/or the cell wall.     

Isoenzyme Patterns in Developing Xanthium Leaves

The isoenzyme patterns of individual Xanthium leaves at various stages of development were determined by acrylamid electrophoresis. The Leaf Plastochron Index was used to measure leaf and plant age. The nature of the changes occurring during leaf development differed from enzyme to enzyme and from gfrom isoenzyme to isoenzyme; for instance, one of the glucose-6-phosphate dehydrogenases was peculiar to very young leaves, another to rapidly expanding leaves, and yet another to still older ones. On the other hand, the number of amylase isoenzymes merely increased with leaf age. Many of the changes in the isoenzyme patterns coincide with the cessation of cell division in the leaf or with the completion of leaf growth. The particular isoenzyme patterns of a given leaf depended on both leaf and plant age. While the isoenzyme patterns of leaves from maximum aldolase activity per unit protein and at a later stage than the leaves from the vegetative plants.     

The occurrence of microbodies and peroxisomal enzymes in achlorophyllous leaves

Summary Several types of leaves of leaf parts lacking chlorophyll were fixed and embedded according to conventional procedures and examined electron-microscopically for microbodies. Comparisons of relative abundance of microbodies, plastids and mitochondria were made by computing the average numbers of organelle profiles per cell section. Similar leaves were homogenized and assayed for three enzymes characteristic of leaf peroxisomes. The localization of these enzymes in microbodies was indicated for the achlorophyllous tissues by the positive result obtained when 3,3-diaminobenzidine was used as an electron cytochemical stain for catalase activity.Microbodies were present in all non-photosynthetic leaves or leaf parts examined, including yellowish-white segments of variegated leaves, albino leaves, and etiolated leaves of two species. In several cases, the numbers of microbody profiles per cell section were as great in the achlorophyllous leaves as in the chlorophyllous. The levels of peroxisomal enzyme activity in the yellowish-white leaves were substantial, although often not as high as in the green leaves. It was concluded that enzymatically these microbodies are probably similar to the peroxisomes characterized from chlorophyllous leaves. In the absence of the photosynthetic product, glycolate, however, it seems unlikely that the organelle is performing the same functions as in green leaves. It is also apparent that the initial formation of peroxisomes in leaves can occur when neither light nor a photosynthate such as glycolate is present as an inducer.     

小麦原生质体分离过程中生理状态的变化

酶解处理使小麦对肉原生质体膜流动性降低,膜脂过氧化产物丙二醛（ＭＤＡ）积累,说明脱璧过程对细胞有伤害作用,损伤位点可能发生在膜上。胚性愈伤组织的具有分裂能力的原生质体,不表现上述变化。酶解脱壁还使超氧化物歧化酶（ＳＯＤ）和过氧化氢酶（ＣＡＴ）活性上升;过氧化物酶（ＰＯＸ）在叶肉原生质体中活性下降,在胚性愈伤组织来源的原生质体中活性上升。以上结果表明：在原生质体分离过程中,细胞的生理特性发生了变化;膜损伤的发生可能与原生质体能否进入正常分裂状态有关。     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demontrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Reversible accumulation of (1-->3,1-->4)-beta-glucan endohydrolase in wheat leaves under sugar depletion.

A (1-->3,1-->4)-beta-D-glucan endohydrolase [(1-->3,1-->4)-beta-glucanase, EC 3.2.1.73] was detected in wheat (Triticum aestivum L.) leaves by Western analyses and activity measurements. This enzyme is able to degrade the (1-->3,1-->4)-beta-glucans present in the cell walls of cereals and other grass species. In wheat, enzyme levels clearly increased during leaf development, reaching maximum values at full expansion and then decreasing upon leaf ageing. To test whether the abundance of (1-->3,1-->4)-beta-glucanase might be controlled by the carbohydrate status, environmental and nutritional conditions capable of altering the leaf soluble sugar contents were used. Both the activity and enzyme protein levels rapidly and markedly increased when mature leaves were depleted of sugars (e.g. during extended dark periods), whereas elevated carbohydrate contents (e.g. following continuous illumination, glucose supply in the dark or nitrogen deficiency during a light/dark cycle) caused a rapid decrease in (1-->3,1-->4)-beta-glucanase abundance or prevented its accumulation in the leaves. The physiological significance of (1-->3,1-->4)-beta-glucanase accumulation under sugar depletion remains to be elucidated.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demonstrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Acid and alkaline invertases in suspension cultures of sugar beet cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K_m values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K_m values for sucrose.     

Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate) aminotransferase in plant tissues

According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis.     

Active, inactive and in vitro synthesized forms of polyphenoloxidase during leaf development

Polyphenoloxidase (PPO; EC 1.14.18.1) activity decreased 8-fold from young to mature Vicia faba L. Moensch (cv. Long pod) leaves. The K_m for catechol remained relatively constant from young to mature leaves. Electrophoretic separation and analysis showed that only one active form was present in extracts from various leaf sizes. The amount of this form appeared to decrease with leaf size/age. In extracts which had not tanned, electroblotting and immunostaining indicated that one enzyme form was present with a molecular mass of 45 kDa. Two leaf categories contained greater amounts of this immunological cross-reacting PPO than other leaf categories. When extracts were allowed to darken, immunoblotting detected three enzyme forms with molecular masses of 45, 59 and 63 kDa. The latter two immunological crossracting species had no detectable enzyme activity. Poly-A⁺ mRNA was isolated from six leaf sizes and translated in vitro. A product corresponding to PPO was present in all leaf categories. Greater amounts of this translation product were observed in medium-sized leaves than in very young or mature leaves. These results suggest that: (1) enzymatic assays for PPO are not reliable indicators of the total amount of PPO protein present in developing leaves, (2) immunoblotting can detect inactive enzyme forms, (3) only one active form of the enzyme is present at all developmental stages, and (4) mRNA corresponding to PPO is present at all developmental stages but appears to be more abundant in certain leaf sizes/ages.     

Subcellular Localization of Proteases in Developing Leaves of Oats (Avena sativa L.)

The distribution and subcellular localization of the two major proteases present in oat (Avena sativa L. cv Victory) leaves was investigated. Both the acidic protease, active at pH 4.5, and the neutral protease, active at pH 7.5, are soluble enzymes; a few percent of the enzyme activity was ionically bound or loosely associated with organellar structures sedimenting at 1000g. On the average, 16% of the acidic protease could be washed out of the intercellular space of the leaf. Since isolated protoplasts contained correspondingly lower activities as compared to crude leaf extracts, part of the acidic activity is associated with cell walls. No neutral protease activity was recovered in intercellular washing fluid. Of the activities present in protoplasts, the acidic protease was localized in the vacuole, whereas the neutral protease was not. The localization of the acidic protease in vacuoles did not change during leaf development up to an advanced stage of senescence, when more than 50% of the leaf protein had been degraded. These observations indicate that protein degradation during leaf senescence is not due to a redistribution of acidic protease activity from the vacuole to the cytoplasm.     

Control of Ribulose-1,5-diphosphate Carboxylase Activity During Expansion of Leaves of Capsicum frutescens L.

The activity of ribulose-1,5-diphosphate carboxylase per unitlaminar area increases rapidly during early stages of leaf expansionin Capsicum frutescens L. cv. California Wonder. This is followedby a decrease to a level that is constant until expansion stops. A previous suggestion that the expansion of younger leaves inthe same phyllotactic sector controlled the decrease in enzymeactivity in older expanding leaves has not been verified byexperiments involving the selective excision of leaves and cotyledons.Decrease in enzyme activity was accompanied by a fall in FractionI protein content but another chloroplastic enzyme, -aminolevulinicacid dehydrase, did not exhibit a decrease in activity. Intra-chloroplasticmechanisms, rather than the influence of other plant organs,are suggested as controlling ribulose-1,5-diphosphate carboxylaseactivity during later stages of leaf expansion.     

Marked modulation by phosphate of phosphoenolpyruvate carboxylase in leaves of Amaranthus hypochondriacus, a NAD-ME type C4 plant: decrease in malate sensitivity but no change in the phosphorylation status

The effect of Pi on the properties of phosphoenolpyruvate carboxylase (PEPC) from Amaranthus hypochondriacus, a NAD-ME type C4 plant, was studied in leaf extracts as well as with purified protein. Efforts were also made to modulate the Pi status of the leaf by feeding leaves with either Pi or mannose. Inclusion of 30 mM Pi during the assay enhanced the enzyme activity in leaf extracts or of purified protein by >2-fold. The effect of Pi on the enzyme purified from dark-adapted leaves was more pronounced than that from light-adapted ones. The Ki for malate increased >2.3-fold and >1.9-fold by Pi in the enzyme purified from dark-adapted leaves and light-adapted leaves, respectively. Pi also induced an almost 50-60% increase in Km for PEP or Ka for glucose-6-phosphate. Feeding the leaves with Pi also increased the activity of PEPC in leaf extracts, while decreasing the malate sensitivity of the enzyme. On the other hand, Pi sequestering by mannose marginally decreased the activity, while markedly suppressing the light activation, of PEPC. There was no change in phosphorylation of PEPC in leaves of A. hypochondriacus due to the feeding of 30 mM Pi. However, feeding with mannose decreased the light-enhanced phosphorylation of PEPC. The marked decrease in malate sensitivity of PEPC with no change in phosphorylation state indicates that the changes induced by Pi are independent of the phosphorylation of PEPC. It is suggested here that Pi is an important factor in regulating PEPC in vivo and could also be used as a tool to analyse the properties of PEPC.     

Nitrogen-deficiency-induced loss in photosynthesis and modulation of β-galactosidase activity during senescence of <Emphasis Type="Italic">Arabidopsis</Emphasis> leaves

Senescence-induced loss in the content of chlorophyll and the rate of oxygen evolution is remarkably enhanced when the leaves of Arabidopsis thaliana experience nitrogen-deficiency stress. On the other hand, the decline in the level of total soluble sugar during senescence is very slow and nitrogen deficiency does not exhibit any further change. The relative stability in the level of the sugar in the background of severe decline of photosynthesis may suggest the contribution of sugars from other sources to sustain its homeostasis to execute and complete energy-dependent senescence process and stress response. The possible participation of cell wall polysaccharides contributing to sugar homeostasis is predicted. Senescence-induced increase in the activity of β-galactosidase (EC 3.2.1.23) and its further enhancement in senescing leaves experiencing nitrogen stress support the proposition of participation of the enzyme for breakdown of the wall polysaccharides to sugars. The loss of photosynthesis as a possible signal for enhancement in the activity of β-galactosidase has been further examined in the excised leaves incubated in Okada and Shimura (OS) nutrient medium with and without nitrogen. Nitrogen limitation experienced by excised leaves causes rapid loss in photosynthesis with concomitant increase in the activity of the enzyme extracted both from soluble and cell wall fractions. The differential activity of the enzyme from soluble and cell wall fractions during development-dependent leaf senescence and premature senescence in excised leaves induced by nitrogen deficiency appears to be complex and needs to be resolved in the future.     

Fine Scale Patterns in Microbial Extracellular Enzyme Activity during Leaf Litter Decomposition in a Stream and its Floodplain

Microorganisms mediate the decomposition of leaf-litter through the release of extracellular enzymes. The surfaces of decomposing leaves are both chemically and physically heterogeneous, and spatial patterns in microbial enzyme activity on the litter surface should provide insights into fine-scale patterns of leaf-litter decomposition. Platanus occidentalis leaves were collected from the floodplain of a third-order stream in northern Mississippi, enclosed in individual litter bags, and placed in the stream channel and in the floodplain. Replicate leaves were collected approximately monthly over a 9-month period and assayed for spatial variation in microbial extracellular enzyme activity and rates of organic matter (OM) decomposition. Spatial variation in enzyme activity was measured by sampling 96 small discs (5-mm diameter) cut from each leaf. Discs were assayed for the activity of enzymes involved in lignin (oxidative enzymes) and cellulose (β-glucosidase, cellobiohydrolase) degradation. Rates of OM loss were greater in the stream than the floodplain. Activities of all enzymes displayed high variability in both environments, with severalfold differences across individual leaves, and replicate leaves varied greatly in their distribution of activities. Geostatistical analysis revealed no clear patterns in spatial distribution of activity over time or among replicates, and replicate leaves were highly variable. These results show that fine-scale spatial heterogeneity occurs on decomposing leaves, but the level of spatial variability varies among individual leaves at the measured spatial scales. This study is the first to use geostatistical analyses to analyze landscape patterns of microbial activity on decomposing leaf litter and in conjunction with studies of the microbial community composition and/or substrate characteristics, should provide key insights into the function of these processes.     

Influence of light on phosphoenol pyravate carboxylase in sorghum leaves

Upon greening of sorghum leaves ( Sorghum vulgare Pers. cv. INRA 450) under white light illumination, phosphoenolpyruvate carboxylase activity increases. 17 times; at the same time, a new isoform of the enzyme appears.
The aim of the present work has been to identify the process responsible for the appearance of this isoform. Greening. of the leaves in the presence of D₂O did not lead to a significant increase in the buoyant density of the enzyme. On the other hand, cycloheximide was a powerful inhibitor of the rise in PEP carboxylase activity. In order to clarify these conflicting data a procedure based on the immunoprecipitation of the enzyme and its quantification by gel electrophoresis was developed in order to estimate the amount of enzyme in leaf tissue. The results clearly demonstrate that light triggers an increased synthesis of the enzyme protein during greening of sorghum leaves.     

Purification and Developmental Analysis of a Metalloendoproteinase from the Leaves of Glycine max

A metalloendoproteinase from leaves of soybean (Glycine max) has been purified 1160-fold to electrophoretic homogeneity. The native protein is monomeric with a molecular mass of 15 kilodaltons as estimated by gel filtration and 19 kilodaltons as estimated by denaturing gel electrophoresis. The enzyme has a pH optima of 8.0 to 9.0 using Azocoll as substrate. The proteolytic activity is susceptible to metal chelating agents and the inactivated enzyme can be restored to 69% of original activity by the addition of ZnCl₂. Western analysis shows that a fraction of the soybean metalloendoproteinase is present within the extracellular space of older leaves. Soybean metalloendoproteinase 1 is the Azocollase A activity first described by Ragster and Chrispeels (Plant Physiol 64: 857-862; 1979).     

Characterization of an extracellular chymostatin-sensitive serine protease preferentially expressed in young plant tissues

Intercellular washing fluids from leaves of all tested higher plant species contained a serine-type protease which efficiently cleaved the artificial fluorogenic substrate MCA-Pro-Leu-Gly-Leu-Dnp-Ala-Arg (MCA). The activity varied between the species. The classification as serine protease was based on the sensitivity towards chymostatin and phenylmethylsulfonyl fluoride. MCA protease activity strongly declined with leaf age and was also detected in stems, roots and flower petals. In tobacco, specific activity of the chymostatin-sensitive MCA protease was about 40-fold higher in intercellular washing fluids than in whole leaf homogenate confirming the extracellular location of the MCA protease. The same enzyme activity was detected in developing tomato fruits; it showed a correlation with fruit growth and was not detectable in ripe fruits. The tobacco protease was sensitive to temperatures above 50 degrees C, had an isoelectric point of 5.8+/-0.1 and an apparent molecular mass of 68 kDa. Its pH optimum was very broad with little difference in activity between pH 5 and 9. Conversely, a casein-cleaving protease also present in intercellular washing fluids was insensitive towards chymostatin and revealed a pronounced pH optimum around 6.0. The data biochemically characterize a new type of extracellular proteolytic activity which may be particularly important during tissue expansion.