Transaminase studies in germinating seeds

The changes in glutamic-alanine and glutamic-aspartic transaminase during the germinating period of the seedling have been studied with plants from three plant families. It was found that in the germinating seed embryo the glutamic-aspartic transaminase activity was in general higher than the glutamic-alanine transaminase activity. At the beginning of germination all plants studied contained approximately the same amount of transaminase: i.e., about 5–10 units/embryo. However after 120 hr. of germination there were wide variations in the amount of transaminase found in the different species. In most of the plants studied the transaminase activity/mg. protein nitrogen revealed a proportionally greater increase in the enzymatic activity than that of the protein nitrogen.Stimulation of growth and metabolism of the embryo by the use of a mineral supplement for germination of the seeds produced an increase in the rate of formation of both transaminase and protein nitrogen. However, the production of the enzyme was relatively greater than the formation of protein nitrogen.The results indicated that no definite relationships existed between the changes in glutamic-alanine or glutamic-aspartic transaminase activity and the formation of protein. This would suggest that in plants, protein synthesis is not a direct function of transaminase activity; it may play an indirect role in protein synthesis by its action on the interconversion of amino and keto acids.     

A Bacterial Indole-3-acetyl-L-aspartic Acid Hydrolase Inhibits Mung Bean (Vigna radiata L.) Seed Germination Through Arginine-rich Intracellular Delivery

Indole-3-acetyl-L-aspartic acid (IAA-Asp) is a natural product in many plant species and plays many important roles in auxin metabolism and plant physiology. IAA-Asp hydrolysis activity is, therefore, believed to affect plant physiology through changes in IAA metabolism in plants. We applied a newly discovered technique, arginine-rich intracellular delivery (AID), to deliver a bacterial IAA-Asp hydrolase into cells of mung bean (Vigna radiata) seeds and measured its effects on mung bean seed germination. IAA-Asp hydrolase inhibited seed germination about 12 h after the enzyme was delivered into cells of mung bean seeds both covalently and noncovalently. Mung bean seed germination was delayed by 36 h when the enzyme protein was noncovalently attached to the AID peptide and longer than 60 h when the enzyme protein was covalently attached to the AID peptide. Root elongation of mung bean plants was inhibited as much as 90% or 80%, respectively, when the IAA-Asp hydrolase was delivered with the AID peptide by covalent or noncovalent association. Further thin-layer chromatography analysis of plant extracts indicated that the levels of IAA increased about 12 h after treatment and reached their peak at 24 h. This result suggests that IAA-Asp hydrolase may increase IAA levels and inhibit seed germination of mung bean plants and that the AID peptide is a new, rapid, and efficient experimental tool to study the in vivo activity of enzymes of interest in plant cells.     

Expression of a Chinese cabbage cysteine proteinase inhibitor, BrCYS1, retards seed germination and plant growth in transgenic Tobacco plant

Phytocystatins are plant cysteine proteinase inhibitors that regulate endogenous and heterologous cysteine proteinases of the papain family. A cDNA encoding the phytocystatin BrCYS1 (Brassica rapa cysteine proteinase inhibitor 1 ) has been isolated from Chinese cabbage (B. rapa subsp.pekinensis) flower buds. In order to explore the role of this inhibitory enzyme, tobacco plants (Nicotiana tabacum L. cv. Samson) containing altered amounts of phytocystatin were generated by over-expressingBrCYS1 cDNA in either the sense or the antisense configuration. The resulting plants hadin vitro enzyme inhibitory activities that were over 10% of those detected in wild type plants. The transgenic plants exhibited retarded seed germination and seedling growth and a reduced seed yield, whereas these properties were enhanced in antisense plants. These data suggest that BrCYS1 participates in the control of seed germination, post-germination and plant growth by regulating cysteine peptidase activity.     

MG2+, K+-DEPENDENT PHOSPHATASE ACTIVITY DURING SEED DEVELOPMENT IN NICOTIANA TABACUM

Cation-dependent phosphatase activity was followed in developing Nicotiana tabacum (tobacco) seeds from pre-pollination to seed maturity. Enzyme activity increased rapidly up to 6 days post-pollination, then dropped off until 13 days after pollination. At 15 days post-pollination, enzyme activity had increased again, and at 17 days post-pollination, it reached its highest level. At 19 days post-pollination (seed maturity) enzyme activity was still 28% higher than that of the pre-pollination stage. Since the two peaks in enzyme activity correlate very highly with endosperm growth (first peak) and embryo growth (second peak), it is suggested that the Mg²⁺, K⁺-dependent phosphatase activity detected in this study is associated with the active transport of nutrients to the endosperm and embryo during seed development. The relatively high amount of enzyme activity remaining in the mature seed may represent a store of transport phosphatase that will be available for immediate use at the time of germination and early seedling growth.     

Growth-Dependent Catalase Localization in Exiguobacterium oxidotolerans T-2-2T Reflected by Catalase Activity of Cells

A psychrotolerant and H₂O₂-resistant bacterium, Exiguobacterium oxidotolerans T-2-2^T, exhibits extraordinary H₂O₂ resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state.     

喜马拉雅紫茉莉根水浸提液对五种植物的化感作用研究

以采自西藏林芝地区的藏药植物喜马拉雅紫茉莉为试材,探讨其根部不同浓度(0.0125、0.025、0.05、0.1 g·mL-1)水浸提液对莴苣、绿豆、小麦、萝卜、油菜等5种植物的化感效应。结果表明,随着浸提液浓度的增加,受试植物的种子萌发及幼苗生长均受到不同程度的抑制,植株体内丙二醛含量持续升高,抗氧化酶活性及叶绿素含量逐渐下降,受试植物不同部位对浸提液敏感程度为地下部分>地上部分。另外,5种植物对浸提液敏感程度为莴苣>绿豆>小麦>萝卜>油菜。     

Ectopic expression of ABSCISIC ACID 2/GLUCOSE INSENSITIVE 1 in Arabidopsis promotes seed dormancy and stress tolerance

Abscisic acid (ABA) is an important phytohormone that plays a critical role in seed development, dormancy, and stress tolerance. 9-cis-Epoxycarotenoid dioxygenase is the key enzyme controlling ABA biosynthesis and stress tolerance. In this study, we investigated the effect of ectopic expression of another ABA biosynthesis gene, ABA2 (or GLUCOSE INSENSITIVE 1 [GIN1]) encoding a short-chain dehydrogenase/reductase in Arabidopsis (Arabidopsis thaliana). We show that ABA2-overexpressing transgenic plants with elevated ABA levels exhibited seed germination delay and more tolerance to salinity than wild type when grown on agar plates and/or in soil. However, the germination delay was abolished in transgenic plants showing ABA levels over 2-fold higher than that of wild type grown on 250 mm NaCl. The data suggest that there are distinct mechanisms underlying ABA-mediated inhibition of seed germination under diverse stress. The ABA-deficient mutant aba2, with a shorter primary root, can be restored to normal root growth by exogenous application of ABA, whereas transgenic plants overexpressing ABA2 showed normal root growth. The data reflect that the basal levels of ABA are essential for maintaining normal primary root elongation. Furthermore, analysis of ABA2 promoter activity with ABA2::beta-glucuronidase transgenic plants revealed that the promoter activity was enhanced by multiple prolonged stresses, such as drought, salinity, cold, and flooding, but not by short-term stress treatments. Coincidently, prolonged drought stress treatment led to the up-regulation of ABA biosynthetic and sugar-related genes. Thus, the data support ABA2 as a late expression gene that might have a fine-tuning function in mediating ABA biosynthesis through primary metabolic changes in response to stress.     

Enzyme Profiles in Seedling Development and the Effect of Itaconate, an Isocitrate Lyase-directed Reagent

Changes in levels of isocitrate lyase, malate synthase, and catalase have been investigated during germination of flax (Linum usitatissimum L.) in the presence and absence of itaconate. Germination was accompanied by a rapid increase in these enzymes during the first 3 days. The presence of 38 millimolar itaconate inhibited the incidence of seed germination and the growth of embryo axes as well as the appearance of isocitrate lyase but did not alter the levels of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase. The specific activity for the latter enzyme was constant throughout germination. Oxalate or succinate, each at 38 millimolar, had no effect upon germination of flax seeds. Itaconate did not inhibit the activities of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase in vitro but was a potent noncompetitive inhibitor of isocitrate lyase (K_i:17 micromolar at 30 C, pH 7.6). Itaconate (at 38 millimolar) did not alter the appearance of malate synthase but reduced the incidence of germination, onset of germination, and growth of the embryo axis as well as the specific activity of isocitrate lyase in seedlings of Zea mays, Vigna glabra, Glycine hispida, Vigna sinensis, Trigonella foenumgraecum, Lens culinaris, and Medicago sativa. The incidence and onset of germination of wheat seeds were unaltered by the same concentration of itaconate but seedlings did not contain isocitrate lyase or malate synthase. The data suggest that itaconate may be isocitrate lyase-directed in inhibiting the germination of fatty seeds.     

Rhizoctonia wilt suppression of brinjal (Solanum melongena L) and plant growth activity by Bacillus BS2

An antibiotic-producing and hydrogen-cyanide-producing rhizobacteria strain Bacillus BS2 showed a wide range of antifungal activity against many Fusarium sp. and brinjal wilt disease pathogen Rhizoctonia solani. Seed bacterization with the strain BS2 promoted seed germination and plant growth in leguminous plants Phaseolus vulgaris and non-leguminous plants Solanum melongena L, Brassica oleracea var. capitata, B. oleraceae var. gongylodes and Lycopersicon esculentum Mill in terms of relative growth rate, shoot height, root length, total biomass production and total chlorophyll content of leaves. Yield of bacterized plants were increased by 10 to 49% compared to uninoculated control plants. Brinjal sapling raised through seed bacterization by the strain BS2 showed a significantly reduced wilt syndrome of brinjal caused by Rhizoctonia solani. Control of wilt disease by the bacterium was clue to the production of antibiotic-like substances, whereas plant growth-promotion was due to the activity of hydrogen cyanide. Root colonization study confirmed that the introduced bacteria colonized the roots and occupied 23-25% of total aerobic bacteria, which was confirmed using dual antibiotic (nalidixic acid and streptomycin sulphate) resistant mutant strain. The results obtained through this investigation suggested the potentiality of the strain BS2 to be used as a plant growth promoter and suppressor of wilt pathogen.     

巨尾桉叶片挥发物对三种植物种子萌发及幼苗生长的化感效应

为探讨人工巨尾桉林叶片挥发物对周边农作物的化感作用,采用不同质量新鲜巨尾桉叶片及由新鲜叶片提取的桉叶油对玉米、辣椒、西红柿等三种植物种子进行处理,观测其种子萌发和幼苗生长状况。结果表明:(1)在三种被测植物中,桉树叶片挥发物对玉米种子萌发影响最小,对辣椒影响最大;(2)当叶片用量小于200g时,桉树叶片挥发物对三种植物种子萌发影响不明显,当叶片用量达400g时,能完全抑制辣椒、西红柿种子萌发,并能极显著降低玉米种子萌发(P0.01);(3)玉米幼苗芽生长随叶片用量的增加呈现先促进后抑制现象,对芽高、鲜重、干重的促进和抑制均达到显著(P0.05)或极显著水平(P0.01);(4)当叶片用量小于或等于100g时,桉树叶片挥发物对辣椒、西红柿幼苗生长影响不明显,当用量达到200g时则能极显著抑制辣椒、西红柿幼苗生长;(5)桉叶油对三种测试植物的抑制效果与叶片自然挥发物相似,且效果更显著。     

Optimized preparation and determination of pea seedling diamine oxidase

The level of diamine oxidase in pea seedling stems has been determined as a function of time after germination in both etiolated and non-etiolated plants. The maximum amount of enzyme per plant is obtained between 11 and 13 days. The amount of activity per gram of tissue appears to be proportional to the rate of growth. We describe an efficient method of isolation of pea seedling stem diamine oxidase from 12-day-old etiolated seedlings, a procedure that brings the enzyme to purity after a 97-fold purification. A new assay procedure for pea seedling diamine oxidase is detailed and compared to previously used methods. The kinetic parameters for three common substrates have also been determined. SDS-acrylamide gel electrophoresis, gel filtration chromatography and copper analyses have been used to determine that pea seedling diamine oxidase exists as a dimer of two apparently identical subunits, the dimer molecular weight being about 190,000. The isoelectric point of this enzyme was determined to be 6.5.     

The emergence of embryos from hard seeds is related to the structure of the cell walls of the micropylar endosperm, and not to endo-beta-mannanase activity

BACKGROUND AND AIMS: Seeds of carob, Chinese senna, date and fenugreek are hard due to thickened endosperm cell walls containing mannan polymers. How the radicle is able penetrate these thickened walls to complete seed germination is not clearly understood. The objective of this study was to determine if radicle emergence is related to the production of endo-beta-mannanase to weaken the mannan-rich cell walls of the surrounding endosperm region, and/or if the endosperm structure itself is such that it is weaker in the region through which the radicle must penetrate. METHODS: Activity of endo-beta-mannanase in the endosperm and embryo was measured using a gel assay during and following germination, and the structure of the endosperm in juxtaposition to the radicle, and surrounding the cotyledons was determined using fixation, sectioning and light microscopy. KEY RESULTS: The activity of endo-beta-mannanase, the major enzyme responsible for galactomannan cell wall weakening increased in activity only after emergence of the radicle from the seed. Thickened cell walls were present in the lateral endosperm in the hard-seeded species studied, but there was little to no thickening in the micropylar endosperm except in date seeds. In this species, a ring of thin cells was visible in the micropylar endosperm and surrounding an operculum which was pushed open by the expanding radicle to complete germination. CONCLUSIONS: The micropylar endosperm presents a lower physical constraint to the completion of germination than the lateral endosperm, and hence its structure is predisposed to permit radicle protrusion.     

Effects of Cellulolytic Ruminal Bacteria and of Cell Extracts on Germination of Euonymus americanus L. Seeds

In past attempts, the experimental germination of the seeds of Euonymus americanus L. in vitro has had little success. However, treatment of seeds with ruminal fluid containing viable microflora has been successful in stimulating germination. In the presence of the cellulolytic ruminal bacterium, Clostridium cellobioparum ATCC 15832, seeds of E. americanus were stimulated to germinate. Subsequent studies were designed to determine whether the bacterium synthesized a cellulolytic enzyme responsible for initiating germination. The cell-free endocellulase from C. cellobioparum induced germination of the seeds. To support the hypothesis that the endocellulase from C. cellobioparum was responsible for triggering germination, a 1,4-beta-d-glucan glucanohydrolase (EC 3.2.1.4) from Penicillum funiculosum was used to treat the seeds. In addition, no germination was obtained from seeds treated with a commercial exocellulase enzyme. Also, Ruminococcus flavefaciens FD-1 was found to initiate germination of E. americanus seeds. Thus, cellulase activity is indicated in the degradation of the testa of the seed, allowing imbibition and germination.     

Optimized Preparation and Determination of Pea Seedling Diamine Oxidase

The level of diamine oxidase in pea seedling stems has been determined as a function of time after germination in both etiolated and non-etiolated plants. The maximum amount of enzyme per plant is obtained between 11 and 13 days. The amount of activity per gram of tissue appears to be proportional to the rate of growth. We describe an efficient method of isolation of pea seedling stem diamine oxidase from 12-day-old etiolated seedlings, a procedure that brings the enzyme to purity after a 97-fold purification. A new assay procedure for pea seedling diamine oxidase is detailed and compared to previously used methods. The kinetic parameters for three common substrates have also been determined. SDS-acrylamide gel electrophoresis, gel filtration chromatography and copper analyses have been used to determine that pea seedling diamine oxidase exists as a dimer of two apparently identical subunits, the dimer molecular weight being about 190,000. The isoelectric point of this enzyme was determined to be 6.5.     

Effect of vitamin C and folic acid on seed vigour response and phenolic-linked antioxidant activity

Folic acid and vitamin C were used in the concentration range of 0-500muM as exogenous growth enhancers to stimulate pea (Pisum sativum) seedling vigour. The results suggest that a concentration of 50muM folic acid and 500muM vitamin C were optimum in maximally enhancing seed vigour and potentially seedling performance according to both agronomic and biochemical seed vigour parameters. Results indicated that germination percentage, shoot weight, shoot height, and root length were enhanced in folic acid and vitamin C treated plants compared to control plants. The levels of enhanced phenolic content in response to folic acid and vitamin C treatments were highest on days 8 and 10. Evaluation of critical biochemical parameters indicated that the average glucose-6-phosphate dehydrogenase (G6PDH) activity and proline content in response to treatments were higher than control and correlated to enhanced phenolic content and DPPH-based antioxidant activity. Key enzymes, guaiacol peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) were also higher in response to treatments and correlated to enhanced phenolic content and DPPH-based antioxidant activity. Taken together, these studies support the hypothesis that the proline-linked pentose phosphate pathway stimulates phenolic synthesis and related free-radical scavenging antioxidant activity. Further, this proline-linked pentose phosphate pathway stimulation in response to folic acid and vitamin C was also correlated to antioxidant enzyme response indicated by the stimulation of GPX, SOD, and CAT activities. Therefore, this study indicates the enhancement of seed vigour response by folic acid and vitamin C as reflected in both agronomic and biochemical responses, and this occurred through the stimulation of phenolic-linked antioxidant response that is likely positively modulated through the proline-linked pentose phosphate pathway.     

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.     

干旱胁迫对五爪金龙种子萌发与幼苗生长的影响

以粤东地区危害严重的入侵杂草五爪金龙为材料,采用聚乙二醇(PEG-6000)模拟不同程度的干旱胁迫,测定了干旱胁迫下五爪金龙种子萌发率、萌发指数及其幼苗的叶绿素含量、细胞水势、生物量、抗氧化酶活性等指标,以探讨五爪金龙的抗旱机理.结果表明:随着干旱胁迫程度的增加,(1)五爪金龙种子的初始萌发时间延迟,萌发率、萌发指数显著降低,在重度干旱胁迫(30%)时,种子完全不能萌发且伴随腐烂现象;(2)五爪金龙幼苗叶绿素含量、细胞水势、生物量及苗高显著降低,根茎比显著增大;(3)幼苗叶片的超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量持续上升,过氧化氢酶(CAT)活性持续下降,过氧化物酶(POD)活性在轻度胁迫时无明显变化,但中度胁迫时显著上升,重度胁迫时显著下降(P<0.05).研究发现,干旱胁迫对五爪金龙种子萌发和幼苗生长均有显著抑制作用,但同时也能通过自身一系列生物学、生理学指标的改变主动适应干旱逆境.     

铁核桃根与叶水浸提液对果园间作绿肥植物的化感作用及其生理机制

以西南地区具有代表性的16种绿肥植物为受体材料,采用培养皿药膜法研究了铁核桃(Juglans sigillata)根水浸提液对受体种子发芽率及幼苗鲜重、干重的化感效应;并进一步研究了铁核桃根、叶水浸提液和胡桃醌对化感效应存在明显差异的4种绿肥植物(绿豆、红三叶、白三叶、花生)种子萌发与幼苗生长以及抗氧化酶特性的影响,以筛选适宜中国西南地区核桃园种植的绿肥植物,探讨核桃根和凋落物对绿肥作物的化感作用机制。结果表明:(1)铁核桃根水浸提液对绿豆的发芽率没有影响,但对绿豆幼苗鲜重和干重有显著抑制作用,而对其他15种绿肥的发芽率和鲜重、干重均有抑制作用。(2)胡桃醌显著抑制绿豆种子萌发,而铁核桃根或叶水浸提液对绿豆种子萌发没有影响。(3)铁核桃根或叶水浸提液以及胡桃醌对绿肥植物幼苗生长的化感效应趋势一致,但核桃根或叶水浸提液的化感效应强于胡桃醌。(4)绿豆幼苗在铁核桃根或叶水浸提液以及胡桃醌处理下,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)的活性均高于其他3种(红三叶、白三叶、花生)受体幼苗,表明绿豆清除活性氧能力高,细胞受损害程度较低,受化感作用影响最弱。研究认为,绿豆为适宜中国西南地区幼龄核桃园种植的间作绿肥植物。