Specific binding of gibberellin A1 to aleurone grain fractions from wheat endosperm

A subcellular fraction enriched in aleurone grains isolatedin glycerol from aleurone layers of wheat endosperm specificallyand reversibly bound GA₁-(³H). Specific binding of GA₁ to otherfractions including spherosomes, nuclei, mitochondria, and plasmamembranes was negligible. The K_d of binding to aleurone grainswas 1.5 µM and the number of specific binding sites 0.45pmoles per mg protein. The presence of Ca⁺⁺ ions was absolutelyrequired for binding. Abscisic acid which inhibits giberellinaction in vivo prevented specific GA₁-binding in vitro. GA₁-bindingto aleurone grains is important to the primary action of thehormone which may involve mobilization of reserves from thealeurone grain-spherosome complex for utilization in membranebiogenesis. ¹ Present address: Section of Cytology, Yale University Schoolof Medicine, New Haven, CT 06510, U.S.A. ³ Present address: Laboratoire de Biologie V?g?tale, Ecole NormaleSup?rieure, 24 rue Lhomond, 75231 Paris, France. (Received March 28, 1977; )     

The osmotic potential of polyethylene glycol 6000

Osmotic potential (ψ_s) of aqueous solutions of polyethylene glycol 6000 (PEG-6000) was curvilinearly related to concentration. At given concentrations, ψ_s increased linearly with temperature. The effects of concentration and temperature on ψ_s of PEG-6000 solutions differ from those for most salts and sugars and apparently are related to structural changes in the PEG polymer. Measurements of ψ_s with thermocouple psychrometers are more negative than those with a vapor pressure osmometer, with the psychrometer probably giving the more nearly correct ψ_s for bulk solutions. An empirical equation permits calculation of ψ_s from known concentrations of PEG-6000 over a temperature range of 15 to 35 C. Viscometery and gravimetric analysis are convenient methods by which the concentrations of PEG-6000 solutions may be measured.     

A Cytochrome c Peroxidase Isolated from Thiobacillus thiooxidans

High phytin containing particles were isolated from rice bran by a combination of a differetial centrifugation and an aqueous two phase system, using dextran 500 and polyethylene glycol 6000. The isolated particles consisted mainly of phytic acid, potassium and magnesium. The chemical composition and electron microscopic observation of the isolated particles confirmed that the electron dense material, which is embedded in aleurone particles of matured rice grains, is potassium and magnesium salts of phytic acid.     

Accumulation of phosphorus, magnesium and potassium in developing rice grains: followed by electron microprobe X-ray analysis focusing on the aleurone layer

The levels of P, Mg and K and their distributions in rice grainswere followed during the ripening period. These elements wererapidly translocated into the grains between the 10th and 20thday after flowering and thence their amounts remained constantuntil maturation. The ratio of the electric charges K⁺+Mg²⁺to –PO₄^2– (monoester-P) became 1 with maturation. Electron microprobe X-ray analysis of transverse sections ofgrains indicated that P and Mg began to concentrate in the aleuronelayer about the 12 th day after flowering. The accumulationprocesses and the distribution patterns of P and Mg were verysimilar throughout the ripening period. In contrast, K was notconcentrated in the aleurone layer until the 19th day and itsdistribution pattern differed from that of P and Mg. (Received July 14, 1978; )     

Radioautographic demonstration of the accumulation site of phytic acid in rice and wheat grains

The distribution of ³H-myo-inositol administered to ripeninggrains of rice and wheat was followed by microradioautography.The ³H was exclusively found in aleurone particles, indicatingthat the accumulation site of phytic acid in monocotyledonousseeds is the aleurone particle. (Received July 25, 1973; )     

Effects of culture medium composition and PEG on hyperhydricity in Dendrobium officinale

Hyperhydricity is a physiological disorder during plant tissue culture that seriously affects regeneration and micropropagation. In this study, Dendrobium officinale plantlets were cultured on solid Murashige and Skoog (MS) medium supplemented with plant growth regulators and various concentrations of sucrose, agar, and polyethylene glycol (PEG)-6000 to explore the effect of osmotic stress on hyperhydricity. The results show that low concentrations of sucrose or agar, as well as PEG-6000 at various concentrations, significantly increase the hyperhydric rate of D. officinale, whereas high concentrations of sucrose or agar did not. Furthermore, high concentrations of PEG-6000 significantly increase total water content, free-water content, relative electrical conductivity, and peroxidase (POD) activity of D. officinale plantlets, whereas they significantly decrease bound-water content, proline content, soluble protein content, soluble sugar content, and superoxide dismutase (SOD) activity. These results indicate that PEG-6000 disrupts the antioxidant system and water metabolism in D. officinale plantlets, as well as increases cell membrane permeability, which might be the key factors for the occurrence of hyperhydricity in this species.

     

用渗透胁迫鉴定小麦种子萌发期抗旱性的方法分析

本以聚乙二醇(PEG)-6000、甘露醇和蔗糖作为渗透剂模拟水分胁迫,胁迫溶液渗透势范围在-0．25MPa到-1．50MPa,分析适于进行小麦种子水分胁迫萌发试验的条件,以鉴定小麦萌发期的抗旱性。结果表明,蔗糖溶液易诱发霉茵,胚芽不能正常生长。渗透势为-0．25MPa的PEG-6000及-0．50MPa的甘露醇胁迫已经显抑制了胚芽伸长;-0．50MPa的PEG-6000及-1．00MPa的甘露醇显抑制种子萌发,随着胁迫强度增加,种子相对发芽率及胚芽长度减小,主要是因为渗透胁迫降低了种子吸水速度,胚芽的相对含水量和渗透势均低。在渗透势相同的胁迫条件下,PEG-6000对小麦种子萌发各项检测值的抑制作用均大于甘露醇。如果目的是通过鉴定小麦种子在高渗溶液中的萌发情况,评价萌发期的抗旱性。选用-0．50MPa的PEG-6000或-1．00MPa的甘露醇较为理想,若同时考虑降低试验成本,则应首选-0．50MPa的PEG-6000。     

2种玉米幼苗耐旱性生理机制研究

以白种皮(白玉米)和黄种皮(黄玉米)2个玉米栽培品种为材料,在水培条件下进行聚乙二醇(PEG-6000)模拟干旱胁迫处理,分析玉米叶片抗旱性相关生理特性和质膜H+-ATP酶活性的变化,探讨2种玉米幼苗耐旱性生理机制。结果表明:(1)在2%、5%、10%PEG-6000处理条件下,随处理浓度和时间的增加,2种玉米幼苗植株失水率上升,叶片蒸腾速率降低,气孔传导率下降;在所有相同处理条件下,白玉米植株失水率明显小于黄玉米,而叶片蒸腾速率和气孔传导率下降幅度明显大于黄玉米,即白玉米的耐旱性比黄玉米强。(2)在相同浓度PEG-6000处理下,白玉米叶片可溶性蛋白、可溶性糖含量、游离脯氨酸含量均高于黄玉米,它在干旱胁迫下的渗透调节能力强于黄玉米。(3)在抗氧化酶体系中,随着PEG-6000胁迫浓度的升高,2种玉米叶片CAT活性呈下降趋势,但白玉米CAT活性在2%和5%PEG-6000胁迫下均显著高于黄玉米,其叶片中H2O2含量显著低于黄玉米。(4)随着PEG-6000胁迫浓度的升高,白玉米叶片质膜H+-ATPase磷酸化水平及其与14-3-3蛋白的结合受到的抑制作用比黄玉米强,白玉米叶片质膜H+-ATPase活性比黄玉米叶片低,叶片气孔开度小于黄玉米,叶片蒸腾速率和气孔传导率均低于黄玉米,这可能是白玉米耐旱性强于黄玉米的一个重要机制。     

Studies on the release of barley aleurone cell proteins: Autoradiography

Summary Both uptake and incorporation of radioactivity from [³H]l-leucine into gibberellic-acid (GA₃)-treated aleurone layers of barley (Hordeum vulgare L.) was enhanced by pretreatment with 5 mM potassium bromate. The effect of 5 mM KBrO₃ on amino-acid incorporation was quantitative rather than qualitative and could be partly reversed by the addition of neutralized casein hydrolysate at 10 mg/ml. Autoradiographs of GA₃-treated aleurone cells pulsed with [³H]leucine showed distribution of silver grains predominantly over the endoplasmic reticulum (ER) and aleurone grains. After chasing with carrier l-leucine for 60 min, fewer silver grains were associated with the ER and aleurone grains while nearly half of the silver was associated with the ground cytoplasm of the cell. Autoradiographs were prepared from aleurone cells previously stratified by ultracentrifugation. After a 10-min pulse of label, the silver grains were found over the central ER zone of centrifuged cells; however, with an increase in duration of the chase, label was found distributed throughout the aleurone grain and spherosome region of the cell. The silver grains which were located over the central zone of centrifuged cells at the end of the pulse were almost exclusively associated with the ER. There is no evidence for association of label with dictyosomes or with vesicles derived from dictyosomes. The experimental evidence indicates that labelled amino acids are incorporated into aleurone cells on the ER and are released from these cells without the participation of a membrane-bound vesicle.     

Dormancy of the barley grain is correlated with gibberellic Acid responsiveness of the isolated aleurone layer

The relationship between barley grain dormancy and gibberellic acid (GA₃) responsiveness of aleurone layers has been investigated. Barley (Hordeum distichum L. cvs Triumph and Kristina) grains were matured under defined conditions in a phytotron. Grains of Triumph plants grown under long-day/warm conditions had lower dormancy levels than grains of plants grown under short-day/cool conditions. Aleurone layers isolated from grains of long-day Triumph plants secreted more α-amylase and had a higher responsiveness to GA₃ as measured by α-amylase secretion. Storage of the grains increased both the percentage of germination and the responsiveness of the aleurone to GA₃. Use of different sterilization methods to break dormancy confirmed the correlation between germination percentage and aleurone layer GA₃ responsiveness. The response of embryoless Triumph grains to GA₃ was lower than that of the isolated aleurone layers, suggesting a role of the starchy endosperm in regulating the GA₃ response of the aleurone layer. Grains of the cultivar Kristina harvested from short day- and long day-grown plants lacked dormancy, and their isolated aleurone layers had a similar responsiveness to GA₃ as measured by α-amylase secretion. The data indicate that the physiological state of the aleurone layers contributes to the percentage germination of the grains.     

Amounts of Nuclear DNA and Internal Morphology of Gibberellin- and Abscisic Acid-deficient Tomato (Lycopersicon esculentum Mill.) Seeds During Maturation, Imbibition and Germination

Using X-ray photography and flow cytometry, the internal morphologyand DNA replication activity of wild type (wt), GA- (gib-1 )and ABA-deficient (sit^w ) tomato (Lycopersicon esculentum Mill.cv. Moneymaker) mutant seeds were studied. During seed formation,from 30 to 45 d after pollination (DAP) the endosperm becomessolid and the seed starts to gain desiccation tolerance. Atthis time significant changes occur in the amounts of DNA inradicle tip cells. At 30 DAP, radicle tip cells of the threegenotypes manifest about 60% of 2C, 30% of 4C and 10% of 8Camounts of DNA. Upon maturation (45 DAP onwards), most cellsin the seeds of the three genotypes arrest in the G₁phase ofthe cell-cycle with 2C amounts of DNA. However, a relativelyhigh proportion of cells with 4C amounts of DNA was detectedin the radicle tip cells ofsit^w compared with wild type andgib-1. At the well-matured stage (60 DAP), there were about 2% ofseeds with free space in wild type andgib-1 , and about 13%insit^w . At the over-matured stage (75 DAP), even more seedswith free space were found insit^w , whereas no increase in theproportion of the seeds with free space was detected in theother two genotypes. In -1.0 MPa PEG-6000 with or without 10µM GA₄₊₇, no germination occurred in well-matured wildtype andgib-1 seeds, whether or not they were dried after harvest.However,sit^w seeds were able to germinate both in over-maturefruit and in -1.0 MPa PEG-6000. Priming of dried seeds in -1.0MPa PEG induced a large amount of free space in almost all seedsof the three genotypes, and nuclear DNA synthesis in the radicletip cells of wild type andsit^w seeds. However, PEG priming offresh (non-dried) seeds had no effect on the amount of freespace and 2C/4C DNA ratios in wild type orgib-1 seeds, but didinduce free space in about 20–25% ofsit^w seeds and provoked4C signals insit^w seeds. Removal of the endosperm and testaopposite the radicle tip of seeds resulted in root protrusion,the induction of free space and an increase of 4C DNA signalsin the three genotypes. It is concluded that ABA is crucialfor the efficient arrest of tomato embryo radicle tip cellsin G₁phase upon maturation, whereas GAs play an important rolein re-initiating 4C DNA levels upon germination. Dormancy; flow cytometry; free space; Lycopersicon esculentum ; maturation; priming; seed; tomato     

彩叶草对模拟干旱胁迫的生理响应

采用盆栽试验,对彩叶草进行PEG-6000浓度为0(对照)、5%、10%、15%、20%(W/V)模拟干旱胁迫,研究在干旱胁迫下彩叶草的生长、渗透调节能力及抗氧化酶活性的变化。结果表明:与对照相比,随着PEG-6000浓度的增加,鲜质量、干质量、含水量、水势、根系脱氢酶活性、无机离子含量包括K+、Na+、Ca2+、Mg2+等均呈下降趋势;NO-3含量呈先下降后上升趋势;硝酸还原酶活性、可溶性蛋白含量、可溶性糖含量、超氧化物歧化酶活性均呈先上升后下降趋势;脯氨酸含量、游离氨基酸含量、过氧化物酶活性、过氧化氢酶活性、超氧阴离子(O-2·)产生速率、质膜透性则呈上升趋势。因此,模拟干旱胁迫对彩叶草生长有抑制作用,且随着PEG-6000浓度增加,其生长受抑制和水分胁迫程度加重;模拟干旱胁迫下,彩叶草不积累K+、Na+、Ca2+、Mg2+和NO-3等无机离子进行渗透调节,而积累脯氨酸、可溶性蛋白、可溶性糖、游离氨基酸等有机小分子物质进行渗透调节,但这4种小分子物质增加幅度不尽相同;轻度模拟干旱胁迫虽增强彩叶草抗氧化酶活性,但仍表现轻度的氧化伤害;重度模拟干旱胁迫加重彩叶草氧化伤害。研究结果可为彩叶草耐旱生理机制的研究积累资料,也为其节水型栽植和养护提供依据。     

Soft perforation of planar bilayer lipid membranes of dipalmitoylphosphatidylcholine at the temperature of the phase transition from the liquid crystalline to the gel state

In contrast to the widely used method of electroporation, the method of soft perforation of lipid bilayers is proposed. It is based on the structural rearrangement of the lipid bilayer formed from disaturated phospholipids at the temperature of the phase transition from the liquid crystalline state to the gel state. This allows us to obtain a lipid pore population without the use of a strong electric field. It is shown that the planar lipid bilayer membrane (pBLM) formed from dipalmitoylphosphatidylcholine in 1 M LiCl aqueous solution exhibits the appearance of up to 50 lipid pores per 1 mm² of membrane surface, with an average single pore conductivity of 31±13 nS. The estimation of a single pore radius carried out with water-soluble poly(ethylene glycol)s (PEGs) showed that the average pore radius ranged between 1.0–1.7 nm. It was found experimentally that PEG-1450, PEG-2000, and PEG-3350 should be in a position to block the single pore conductivity completely, while PEG-6000 fully restored the ionic conductivity. The similarity of these PEG effects to ionic conductivity in protein pores makes it possible to suggest that the partition of the PEG molecules between the pore and the bulk solution does not depend on the nature of the chemical groups located in the pore wall.     

Effect of PEG-6000 imposed drought stress on RNA content,relative water content (RWC), and chlorophyll content in peanut leaves and roots

Drought, one of the environmental stresses, plays crucial role in reduction in plant production on majority of agricultural fields of world, In order to evaluate drought stress on RNA content Relative water content (RWC), and chlorophyll content, Water deficit was induced by Polyethylene glycol (PEG) in peanut (Arachis hypogaea), accession number ICGV 91114. In this current study we evaluate RNA content and Relative water content (RWC) both in leaves and roots and chlorophyll content in leaf. The present study was undertaken with the aim to investigate the effect of water deficit imposed by PEG-6000, 40 old day seedlings were treated with varying concentrations of polyethylene glycol-6000 (PEG-6000; w/v-5%, 10%, 15% & 20%) for 24 h. The results showed that RNA content and Relative water content (RWC) content was significantly reduced in both leaves and roots with increased concentration of PEG, In leaves, a concentration dependent decline in chlorophyll content with increasing concentration of polyethylene glycol-6000 (PEG-6000). Reduction in chlorophyll ‘a’ level was to a greater extent than the chlorophyll ‘b’. Thus, this attributes can be used as screening tool for drought tolerance in peanut.     

A simple procedure to overcome polyethelene glycol toxicity on whole plants

A procedure is described that can be used to minimize toxic effects of polyethylene glycol (PEG) to plants. The procedure is based on recycling nutrient solutions containing PEG-6000 through two plant cultures. Tomato plants grown in −0.3 megapascals PEG solutions used after two growth cycles exhibited minimal toxic effects. Long-term responses like dry matter production and chlorophyll content as well as short-term responses like CO₂ fixation rates and leaf conductance were severely inhibited by fresh PEG-6000 and only slightly reduced by recycled PEG-6000. Complete osmotic adjustment was obtained with tomatoes grown in recycled but not in fresh PEG solutions.     

The maize disorganized aleurone layer 1 and 2 ( dil1, dil2) mutants lack control of the mitotic division plane in the aleurone layer of developing endosperm

The maize (Zea mays L.) endosperm consists of an epidermal like layer of isodiametric aleurone cells surrounding a central body of starchy endosperm cells. In disorgal1 (dil1) and disorgal2 (dil2) mutants the control of the mitotic division plane is relaxed or missing, resulting in mature grains with disorganized aleurone layers. In addition to orientation of the division plane, both the shape and size of the aleurone cells are affected, and often more than one layer of aleurone cells is present. Homozygous dil1 and dil2 grains are shrunken due to reduced accumulation of starchy endosperm and premature developmental arrest of the embryo, and mature mutant grains germinate at a very low rate and fail to develop into plants. However, homozygous mutant plants can be obtained through embryo rescue, revealing that both mutants have an irregular leaf epidermis as well as roots with a strongly reduced number of root hairs and aberrant root hair morphology. Our results suggest the presence of common regulatory mechanisms for the control of cell division orientation in the aleurone and plant epidermis.Abbreviations DAP days after pollination - dek defective kernel mutant - dil disorganized aleurone layer mutant - GUS -glucuronidase - LM light microscopy - PPB pre-prophase band - SEM scanning electron microscopy - TUSC Trait Utility System for Corn     

Gibberellic acid and the fine structure of barley aleurone cells

Summary This paper describes changes in the fine structure of barley aleurone cells following treatment with gibberellic acid (GA₃). Within 2 hr of GA₃ treatment the aleurone grains lose the spherical appearance characteristic of aleurone cells incubated in water and buffer alone. This swelling increases with increased exposure of the cells to GA₃ and reaches a maximum at about 10 hr. Accompanying this increase in volume of the aleurone grains is an increase in the amount of rough endoplasmic reticulum. The relevance of these GA₃-stimulated changes in aleurone-cell fine-structure to GA₃-regulated -amylase production is discussed.Work supported by National Science Foundation grants GB-5863 and GB-8332. The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged.     

ULTRASTRUCTURE OF THE MATURE UNGERMINATED RICE (ORYZA SATIVA) CARYOPSIS. THE CARYOPSIS COAT AND THE ALEURONE CELLS

The results of a light and electron microscopic study of the caryopsis coat and aleurone cells in ungerminated, unimbibed rice (Oryza sativa) caryopses are presented. Surrounding the rice grain is the caryopsis coat composed of the pericarp, seed coat and nucellar layers. The outermost layer, the pericarp, consists of crushed cells and is about 10 μm thick. The seed coat, interior to the pericarp, is one cell thick and has a thick cuticle. Between the seed coat cuticle and endosperm are the remains of the nucellus. The nucellus is about 2.5 μm thick and has a thick cuticle adjacent to the seed coat cuticle. Interior to the caryopsis coat is the aleurone layer of the endosperm. The aleurone completely surrounds the rice grain and is composed of two cell types—aleurone cells that surround the starchy endosperm and modified aleurone cells that surround the germ. The aleurone cells of the starchy endosperm contain many aleurone grains and lipid bodies around a centrally located nucleus. The modified aleurone cells lack aleurone grains, have fewer lipid bodies than the other aleurone cells, and contain filament bundles (fibrils). Plastids of aleurone cells exhibit a unique morphology in which the outer membranes invaginate to form tubules and vesicles within the plastid. Transfer aleurone cells are not observed in the mature rice caryopsis.     

Effect of Gibberellic Acid on the Ultrastructure and {alpha}-Amylase Activity of Aleurone Cells of Avena sativa L.

The -amylase activity and ultrastructure of aleurone cells inseeds of Avena sativa L. were studied using seed halves withembryo (embryo seeds) which had imbibed water and seed halveswithout embryo (embryo-less seeds) which had imbibed water withor without GA₃. -Amylase activity was detected in the aleurone layers of embryoseeds that had imbibed water and embryo-less seeds that hadimbibed GA₃-water. The ultrastructure of aleurone cells withdetectable -amylase activity showed marked changes in the roughsurfaced endoplasmic reticulum (rER), in the flattened sacculesforming stacks and in the aleurone grains. The progressive changesin the rER were as follows: first, the number of slender rERincreased; then, the inner space became wider and showed roundor oval profile; and finally, the rER became slender again witha reduced number of adhering ribosomes. The flattened sacculesforming stacks were appressed to the surface of aleurone grains.With time, they decreased in number and finally disappeared.In parallel with the decrease of flattened saccules, digestionof proteinaceous material inside the aleurone grains proceeded. (Received February 24, 1987; Accepted September 3, 1987)