Purification and Properties of a Ribonuclease from Cowpea Cotyledons

The isolation and characterisation of cotyledonary ribonucleases (RNase; EC 3.1.27.1), are basic steps to understand the physiology and biochemistry of RNA turnover and mobilisation during seed germination and seedling establishment, as well as how environmental stresses affect them. RNase was isolated and purified 928-fold, to apparent electrophoretic homogeneity from 5-d-old seedlings of Vigna unguiculata. It is a protein with an apparent molecular mass of 16 kDa having three major isoforms. Its optimum pH is 5.8, which decreases to 5.2 in presence of KCl. It has an apparent K_m of 0.80 mg RNA cm^-3 and retains 40 % of its activity when heated to 80 °C. It is completely inhibited by Cu²⁺, Hg²⁺ and Zn²⁺ and is almost insensitive to Mg²⁺, Ca^2+- and EDTA. Urea, Fe²⁺, Co²⁺ and 2-mercaptoethanol partially inhibit its activity. Its amino acid composition shows a resem lance to that of other plant RNases. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and properties of Pinus taeda arginase from germinated seedlings

In germinated loblolly pine (Pinus taeda L.) seeds arginine accumulates in the seedling during its growth immediately following germination. The enzyme arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is responsible for hydrolyzing this arginine into ornithine and urea. Loblolly pine arginase was purified to homogeneity from seedling cotyledons by chromatographic separation on DE-52 cellulose, Matrex Green and arginine-linked Sepharose 4B. The enzyme was purified 148-fold and a single polypeptide band was identified as arginase. The molecular mass was determined to be 140 kDa by FPLC, while the subunit size was shown to be 37 kDa by SDS-PAGE, predicting a homotetramer holoprotein. Removal of manganese from the enzyme abolishes catalytic activity, which can be restored by incubating the protein with Mn²⁺. Antibodies, raised against the arginase subunit, are able to immunotitrate arginase activity and are monospecific for arginase on immunoblots.     

Metabolism of Inositol Phosphates: I. Phytase Synthesis during Germination in Cotyledons of Mung Beans, Phaseolus aureus

The degradation of phytin in germinating mung bean seeds has been found to be associated with the increased activity of phytase in the cotyledon. In the differentiated embryo the increase of this activity is very low all throughout the growth periods studied. Phytase appears in the cotyledon during germination. No activity has been detected in the cotyledons of unsoaked seeds. Cycloheximide (10⁻⁶ M) inhibits the appearance of phytase by 61% during 24 and 48 hours after the start of germination. This phytase increase is dependent on the synthesis of new RNA in the cotyledon. Synthesis of DNA is not detected in the cotyledon during germination.     

Biochemical characterization of the RNA-hydrolytic activity of a pumpkin 2S albumin

A pumpkin 2S albumin with ribonuclease (RNase) activity was purified from pumpkin seeds (Cucurbita sp.) by liquid chromatographic techniques. It manifested potent RNase activity toward baker’s yeast RNA and calf liver RNA, and some polyhomoribonucleotides, including poly(A), poly(U) and poly(C) but not poly(G). Moreover, it was able to hydrolyze total RNA of both animal and plant origins. Ions such as Na⁺, Mg²⁺, Ca²⁺, and Zn²⁺ inhibited its RNase activity. Since RNase activity has not been previously reported in 2S albumins, this work may shed further light on the biological importance of this group of proteins.     

Halophyte and glycophyte salt tolerance at germination and the establishment of halophyte shrubs in saline environments

Saline sites suffer variations in surface salinity, available soil water, temperature, soil crust strength and other factors which can influence germination and establishment. For establishment to occur the germinating seed must capitalise on a window of opportunity. This window can be widened by placing seeds in a low-salt niche, covering the seeds with a mulch (such as vermiculite), spraying the seed and mulch placement with a coating which may stabilise the favourable situation and raise soil temperature. In this paper it is shown that using seeds collected from plants of Atriplex amnicola which produce many volunteer seedlings in their vicinity can assist establishment from direct seeding. These seeds had the ability to germinate under saltier and cooler conditions than seeds from A. amnicola bushes which did not produce volunteers. Seeds of a halophyte (Atriplex lentiformis) and a non-halophyte (Medicago sativa) are able to imbibe water from a saline substrate in a similar manner. The water enables the seeds of both species to mobilise stored growth materials and produce and elongate radicles. When the seedlings try to erect a hypocotyl and spread their cotyledons, the non-halophyte, in a saline medium, becomes flaccid, distorted and dies. The halophyte seedling shows evidence of high salt tolerance in the form of succulence of cotyledons and trichomes on true leaves even before they are visible and goes on to successfully develop a functioning plant. Nevertheless, germination of halophyte seeds is inhibited or severely reduced at salinity levels above 250 mM NaCl and slowed and reduced progressively up to those levels.     

The role of cotyledons in the establishment of Suaeda physophora seedlings

The role of cotyledons in seedling establishment of the euhalophyte Suaeda physophora under non-saline and saline conditions (addition of 1 mM or 400 mM NaCl) was investigated. Survival and fresh and dry weights were greater for seedlings grown in the light (12-h light/12-h dark) than in the dark (24-h dark). The shading of cotyledons tended to decrease shoot height, shoot organic dry weight, number of leaves, and survival of seedlings regardless of NaCl treatment, but the effect of cotyledon shading was greater with 400 mM NaCl. Concentrations of Na⁺ were higher in cotyledons than in leaves, regardless of NaCl treatment. The K⁺/Na⁺ ratio was lower in cotyledons than in leaves for seedlings treated with 1 mM NaCl but not for seedlings treated with 400 mM NaCl. Addition of 400 mM NaCl decreased oxygen production in cotyledons but especially in leaves. These results are consistent with the hypothesis that, by generating oxygen via photosynthesis and by compartmentalizing Na⁺, cotyledons are crucial for the establishment of S. physophora seedlings in saline environments.     

Gibberellin A3 reverses the effect of salt stress in chickpea (Cicer arietinum L.) seedlings by enhancing amylase activity and mobilization of starch in cotyledons

The percentage germination of chickpea seeds (Cicer arietinum L.cv. PBG-1) gradually decreased with increasing concentration of NaCl in the growth medium and was completely inhibited with 200 mM NaCl. In the presence of 75 mM NaCl, only 51% of the seeds germinated. Gibberellic acid (GA3) and kinetin at 6 µM concentration induced the maximum increase in % germination and seedling growth under salt stress. However, IAA further inhibited both the germination and growth of stressed seedlings. The reduction in amylase activity in cotyledons of stressed seedlings was partially reversed with GA3 and kinetin whereas IAA did not show any positive effect. GA3 was more effective than kinetin in enhancing the reduced germination and seedling growth of chickpea seeds along with amylase activity in cotyledons under NaCl induced saline conditions. The reduced uptake of radiolabelled 14C sucrose by cotyledons and its reduced distribution in the shoots and roots of stressed seedlings was increased with addition of GA3 in the medium. Cotyledonary amylase was separated into amylase 1 and amylase 2 by sephadex G 150 column chromatography. The reduced activities of both amylase 1 and amylase 2 in cotyledons under salt stress was returned to near normal levels with GA3 and there was also an increase in starch utilization, resulting in its lower concentration in cotyledons of GA3-supplemented stressed cotyledons.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

Adenosylhomocysteinase and adenosine nucleosidase activities in Lupinus luteus cotyledons during seed formation and germination

The activities of adenosylhomocysteinase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) were assayed in extracts from yellow lupin (Lupinus luteus L.) cotyledons at different stages of seed formation and seedling development. Adenosylhomocysteinase activity was demonstrated in all the cotyledon extracts examined. Its lowest level was found in the dry seeds and the highest, in 4-day-old seedling cotyledons. Extracts from the cotyledons of maturating seeds, dry seeds, and seedlings up to the second day of growth exhibited no adenosine nucleosidase activity. Adenosine nucleosidase activity appeared in the cotyledons of 2-day-old seedlings and its highest level was reached in 4-to 5-day-old seedlings. There is no inhibitor of adenosine nucleosidase in the maturating and dry yellow lupin seeds. No activator of a possible zymogen form of adenosine nucleosidase from maturating or dry seeds occurs in the growing seedlings.     

RNase and DNase activities in the alfalfa and lentil grown in iso-osmotic solutions of NaCl and mannitol

Nucleolytic activities from two plants of Leguminosae family were determined in order to consider if the nucleases of plants which belong to the same family or to the same species responded in similar ways to stress conditions during growth. Growth parameters of both plants were examined in parallel. In detail, seedlings from two plants, alfalfa (Medicago sativa L. cv. Luzerne Euver) and lentil (Lens culinaris cv. Thessalia), showed significant differences in response to iso-osmotic solutions of NaCl (100 mmol · L⁻¹ solution equivalent to conductivity 8.0 dS m⁻¹) and mannitol (190 mmol · kg⁻¹). Plant height and dry weight of mannitol/NaCl-treated seeds in both plants were lower in comparison to controls (water). Mannitol stress reduced height and dry weight in alfalfa seedlings more than did NaCl. By contrast, lentil seedling growth was inhibited more by NaCl stress than mannitol. In addition, DNase and RNase response to mannitol stress differed in each plant compared to the controls. Mannitol stress induced a sharp increase in DNase- and RNase-specific activity during the initial stages of alfalfa seedlings' growth, followed by a decrease during subsequent days; in lentil seedlings, these activities were inhibited throughout the entire growth period. NaCl stress inhibited the above activities in both plants. After native electrophoresis on gels polymerized in the presence of DNA/RNA, the overall band intensities confirmed the above quantitative results of alfalfa RNase and DNase activity. In addition, the active gel analysis revealed that the decrease of nucleolytic activities in mannitol-treated alfalfa seedlings was mainly due to the strong reduction of acid nucleases. This is the first report of different non-ionic osmotic response of type I plant nucleases during seedlings' growth. In vitro, the addition of up to 300 mmol/L mannitol did not affect acid and neutral nuclease activity in enzyme preparations extracted, purified, and separated from control and mannitol-treated alfalfa seedlings.Our results suggest that plant nucleases responded in a different way to osmotic stress and ionic stress conditions during seedlings' growth.     

Chlorophyll in desiccated seeds of a euhalophyte, Suaeda physophora, and its significancy in plant adaptation to salinity during germination

The seed cotyledons of a euhalophyte, Suaeda physophora, were found to be dark green. The pigment extracted from the cotyledons was proved to be chlorophyll for the absorption spectra curve of extracts the same as that for leaves. Photosynthetic oxygen-exchanging could be detected after the seeds were moistened for 6 h. Microstructure of organelles in cotyledons of ungerminated seeds was detected by transmission electron microscopy. Histochemical pigmentation was used to investigate the degree of damage on the membrane of radicles. A xerophyte, Haloxylon persicum, whose cotyledons of desiccated seeds also contain chlorophyll was used as a comparative species. The results showed that S. physophora maintained the ultra-structure of chloroplasts, the integrity of plasma membranes of radicles kept much better than that of H. persicum, which showed the great adaptability to salinity of the euhalophyte even at the seed-germination stage. Seeds were incubated in 0 and 700 mmol/L NaCl for 10 days in darkness at 20 ℃ , then ungerminated seeds in NaCl solution were transferred to deionised water and reincubated for another 8 days and was recorded the germination recovery. Dry seeds moistened with deionised water germinated gradually in the dark and germination was maximal after 3 or 4 d. Seeds at 700 mmol/L NaCl for 10 d and transferred back to deionised water germinated abruptly, most seeds germinated in one day and photosynthesis was also detected. It is concluded that recovery germination of S. physophora ensured the seeds could germinate rapidly after salinity declines, for example under the mild but transitory favorable edaphic condition in early spring. Photosynthesis function in seeds might also promoted radicle growth and seedling establishment.     

外源水杨酸对盐胁迫下大黄种子萌发及幼苗生长的影响

该研究以掌叶大黄、唐古特大黄和药用大黄种子为材料,采用双层滤纸培养法,设置系列浓度NaCl (0、100、150、200、250 mmol/L) 胁迫试验,以及系列浓度水杨酸(SA)溶液(0、50、100、150、200、250 mg/L)拌种和浸种后盐胁迫实验,测定3种大黄种子萌发及幼苗生长指标,揭示外源水杨酸对盐胁迫下大黄种子萌发及幼苗生长的影响。结果显示：(1)随NaCl浓度增大3种大黄种子的发芽率均呈直线下降趋势,且子叶、胚轴、根和苗等生长均受到强烈抑制。(2)在拌种条件下, 200 mmol/L NaCl胁迫下掌叶大黄苗长在200 mg/L SA处理下受到显著促进; 200 mmol/L NaCl浓度盐胁迫下唐古特大黄种子发芽率在250 mg/L SA处理下受到显著抑制;100 mmol/L NaCl胁迫下药用大黄种子发芽势在200 mg/L SA处理下受到显著抑制,其发芽率在150 mg/L SA处理下得到显著抑制,其苗长在250 mg/L SA处理下受到显著抑制。(3)在浸种条件下, 200 mmol/L NaCl胁迫下掌叶大黄种子发芽率在50 mg/L SA处理下显著提高,其幼苗根长和苗长的生长在250 mg/L SA处理受到显著促进;200 mmol/L NaCl胁迫下唐古特大黄种子的发芽势在200 mg/L SA处理下得到显著促进,其幼苗根和苗的生长在50 mg/L SA处理下得到显著促进;100 mmol/L NaCl 胁迫下药用大黄根和苗的生长在100 mg/L SA处理下均得到显著促进。研究表明,3种大黄种子和幼苗对盐胁迫的响应趋势一致,但对不同浓度SA拌种和浸种的响应有较大差异。     

Gibberellin-mediated changes in carbohydrate metabolism during flower stalk elongation in tulips

We tested the effects of cold stratification, temperature, light and NaCl on seed germination and germination recovery and of NaCl on radicle growth and radicle elongation recovery of Kalidium caspicum, a small leafy succulent shrub dominant in saline deserts in northwest China. In all conditions of temperature and light/darkness, germination percentages and rates of cold-stratified seeds were significantly higher than those of nonstratified seeds. Germination of a high percentage of both nonstratified and stratified seeds was inhibited by 0.2 M NaCl, and 0.6 M NaCl completely inhibited germination. Nongerminated seeds germinated after they were transferred from NaCl solutions to distilled water. Radicle elongation significantly decreased with increase in salinity, and it was completely inhibited by ≥1.0 M NaCl; radicle elongation recovered in young seedlings pretreated by 10 days of incubation in ≤0.4 M NaCl. Results show that seed germination and early seedling growth of K. caspicum are salt tolerant, and these characteristics help explain why this species can survive and dominate salt habitats, such as those in the Junggar desert in Xinjiang, northwest China.     

Untersuchungen über rnasen in kotyledonen von nachgereiften und dormantenagrostemma githago-samen

Activities of RNasea were studied in cotyledons of dormant and afterripenedAgrostemma githago seeds. Activity of RNase increases during imbibition and germination. This increase in activity cannot be observed in variants which are not able to germinate (dormant seeds and seeds blocked by higher temperature). The development of RNase activities during germination cannot be inhibited by concentrations of cycloheximide or actinomycine D completely preventing phosphatase synthesis. These results may be indicative for the assumption that the increase of RNase during germination is caused by enzyme activation and not by enzyme synthesis. Cytokinins and a combination of cycloheximide and gibberellic acid stimulate the activity of RNase in dormant cotyledons, whereas neither cycloheximide nor gibberellic acid, applicated by themselves, show any effect. Cytokinins and gibberellic acid do not influence the activity of RNase of afterripened cotyledons, abscisic acid inhibits the increase of enzyme activity. There are characteristic changes in the pattern of RNases during germination revealed by polyacrylamide gel electrophoresis. The increase in RNase activity of dormant cotyledons caused by cytokinins is accompanied by obvious changes in the RNase pattern on polyacrylamide gel. Treating dormant cotyledons with cytokinins dormancy is partially overcome. In consequence of the application of cytokinins the differences in the electrophoretic RNase pattern between dormant and afterripened cotyledons can be nearly balanced.     

Nucleases in chloroplast of barley

Two barley chloroplast nuclease fractions were separated by the affinity chromatography and gel electrophoresis. Both were about 2 times more active to RNA than to native DNA and about half as active to denaturated DNA as to native DNA. Both fractions were as active to UV-irradiated (270 J m^-2) native DNA as to intact DNA but their action was inhibited by apurinic sites. The enzyme activities were inhibited by high concentrations of EDTA, NaCl, Mn²⁺, Ca²⁺, Zn²⁺ ions and by N-ethylmaleimide. They do not require Mg²⁺ ions but are stimulated or at higher concentration inhibited by their presence. Both RNase and DNase were active over a wide pH range (5.5–9), the optimum for DNase action in the presence of Mg²⁺ being 6.5, for RNA decomposing activity at pH 8.0. As no mononucleotides were detected in acid soluble form, it seems likely that DNase acts in the endonucleolytic way.     

Carbamoyl phosphate synthetase activity from the cotyledons of developing and germinating pea seeds

Carbamoyl phosphate synthetase activity was measured in partially purified extracts from cotyledons of developing and germinating seeds of Pisum sativum L. Some properties of the enzyme were established. During cotyledon development, the activity initially increased sharply but decreased during further development. The activity from germinating seeds was only one-tenth of the maximum activity at an early developmental phase. The results are discussed in relation to pea seed development and germination.     

盐磷胁迫对木麻黄和台湾相思种子萌发及幼苗生长的影响

恶劣环境下,人工海防林因面临养分胁迫而经营困难。为探讨盐、磷胁迫对主要海防林树种木麻黄和台湾相思种子萌发及生长的影响,该研究分别用不同浓度的NaCl(盐)和KH₂PO₄(磷)溶液处理种子和浇灌幼苗,测定种子萌发和幼苗生长指标。结果表明：(1)高盐胁迫显著抑制种子萌发,对幼苗生长有一定影响,但两种植物影响程度不同;台湾相思种子萌发耐盐性高于木麻黄,前者相对盐害率最大值为23.03%,后者为89.15%;随着盐浓度增加,木麻黄和台湾相思种子的发芽率、发芽势、发芽指数和活力指数均降低,对应最大值分别为38.70%、34.67%、18.70、0.055和76.67%、62.22%、48.46、6.11。(2)两种植物的株高和根长随盐浓度增加而降低,木麻黄和台湾相思株高分别为12.29～6.01 mm和48.27～17.33 mm,根长分别为8.57～1.45 mm和33.41～5.88 mm;台湾相思根、茎、叶生物量及根冠比均随盐浓度的增加逐渐减小,木麻黄各处理差异较小。(3)台湾相思的种子和幼苗较木麻黄更耐低磷环境,二者最适磷浓度存在差异;木麻黄种...     

Mitochondrial malate dehydrogenase of watermelon cotyledons: Time course and mode of enzyme activity changes during germination

Summary Specific antibodies were prepared against the purified mitochondrial malate dehydrogenase (EC 1.1.1.37) from cotyledons of watermelon seedlings (Citrullus vulgaris Schrad.). The isoenzyme was assayed by means of quantitative radial immunodiffusion. Cotyledons of ungerminated seeds were found to contain mitochondrial MDH. During the first 4 days of germination the enzyme activity increased threefold finally contributing 16% to the total MDH activity extracted from cotyledon tissue. Isopycnic CsCl density centrifugation was used to investigate the mode of activity increase. After a four-day period of labelling with deuterium oxide and purification of the mitochondrial isoenzyme, a density shift of 0.021kgx1^-1, accompanied by considerable band broadening of the enzyme profile was observed. These findings are evidence for the de novo synthesis of mitochondrial MDH and its relatively slow turnover in germinating seeds.Abbreviations mMDH mitochondrial malate dehydrogenase - D₂O deuterium oxide     

Initiation of the degradation of the soybean kunitz and bowman-birk trypsin inhibitors by a cysteine protease

Protease K1 activity initiates the degradation of the Kunitz soybean trypsin inhibitor (KSTI) during germination and early seedling growth. This enzyme was purified nearly 1300-fold from the cotyledons of 4-day-old soybean (Glycine max [L.] Merrill) seedlings. Protease K1 is a cysteine protease with a molecular weight of approximately 29,000. It cleaves the native form of KSTI, Ti^a, to Ti^a_m, the same modified form observed in vivo. In addition to attacking KSTI, protease K1 is also active toward the major Bowman-Birk soybean trypsin inhibitor, as well as the α, α′, and β subunits of soybean β-conglycinin. The properties and temporal variation of protease K1 during germination indicate that it is responsible for initiating the degradation of both KSTI and Bowman-Birk soybean trypsin inhibitor in the soybean cotyledon.     

Inhibition of ribonuclease and protease activities in germinating rice seeds exposed to nickel

When the seeds of two rice cvs. Malviya-36 and Pant-12 were germinated up to 120 h in the presence of 200 and 400 μM NiSO₄, a significant reduction in the germination of seeds occurred. Seeds germinating in the presence of 400 μM NiSO₄ showed about 12–20% decline in germination percent, about 20–53% decline in lengths and about 8–34% decline in fresh weights of roots and shoots at 120 h of germination. Ni²⁺ exposure of germinating seeds resulted in apparent increased levels of RNA, soluble proteins, and free amino acids in endosperms as well as embryo axes. A 400 μM Ni²⁺ treatment led to about 58–101% increase in the level of soluble proteins and about 39–107% increase in the level of free amino acids in embryo axes at 96 h of germination. Activities of ribonuclease and protease declined significantly with increasing levels of Ni²⁺ treatment. Isoenzyme profile of RNase as revealed by activity staining indicated decline in the intensities of 3–4 preexisting enzyme isoforms in embryo axes of both the rice cultivars and disappearance of one of the two isoforms in endosperms of cv. Pant-12 due to 400 μM Ni²⁺ treatment. Results suggest that the presence of high level of Ni²⁺ in the medium of germinating rice seeds serves as a stress factor resulting in decreased hydrolysis as well as delayed mobilization of endospermic RNA and protein reserves and causing imbalance in the level of biomolecules like RNA, proteins, and amino acids in growing embryo axes. These events would ultimately contribute to decreased germination of rice seeds in high Ni²⁺ containing environment.