DON抑制小麦种胚细胞周期启动的研究

小麦种子在０．５－４．０ｍｇ／Ｌ的脱氧雪腐镰刀菌烯醇（ＤＯＮ）中溶液中萌发,用流式细胞仪检测胚细胞的周期时相并观察黄化苗的生长。结果表明,对照组胚细胞在萌发１２ｈ开始启动细胞周期,此后Ｓ期细胞比率迅速增加并在２０ｈ达到最大值,较萌发１０ｈ的６．５％增加１．７８倍,约１４％的胚细胞在萌发的２２－２６ｈ间完成第一次分裂;ＤＯＮ抑制Ｇ１期胚细胞的启动,小麦胚中Ｓ期细胞比率随ＤＯＮ浓度增加呈指数下降,３．     

超声波辅助农杆菌介导的小麦遗传转化初步研究

报道了一种改进的小麦转化方法——超声波辅助的农杆菌转化法。发芽的小麦种子经超声波处理20min后再经农杆菌浸染,浸染后的种子按正常播种和栽培管理至结实,对收获的成熟种子进行卡那霉素抗性筛选。结果表明,受试的4个小麦品种均得到抗性后代,PCR扩增结果证明这些抗性植株是转化植株。GUS染色结果表明不经超声波处理的种子几乎看不到转化的细胞,但经过超声波处理的种子,幼苗基部的细胞转化成功。研究表明,利用超声波辅助的农杆菌转化方法转化小麦能产生可遗传的后代。     

The effects of endogenous cytokinin content on benzyladenineenhanced nitrate reductase induction

Foliar application of benzyladenine (BA) has been shown to enhance nitrate-dependent induction of nitrate reductase (NR; EC 1.6.6.1) in etiolated wheat ( Triticum aestivum L.) seedlings. Whether similar enhancement occurs in light-grown plants, or whether endogenous cytokinin content affects this enhancement is unknown. Since the cytokinin content of etiolated plants probably differs from that of light-grown seedlings, the NR response of each to exogenous root- or shoot-applied BA in wheat (cv. Red Bob) was examined. Endogenous cytokinins present in untreated control tissues prior to BA application and changes that occurred after a 22 h (12 h dark followed by 10 h of light) period were determined using a combined HPLC-immunoassay method. Shoot application of BA enhanced the induction of NR in etiolated seedlings in a concentration-dependent manner but failed to enhance NR induction in light-grown plants. Root-applied BA enhanced NR induction in both etiolated and light-grown seedlings. Endogenous root cytokinin levels were similar in both etiolated and light-grown plants. In contrast, shoots of 6 day-old light-grown seedlings contained at least 20 times the amount of total cytokinins measured in shoots from etiolated plants of the same age. Total cytokinin content of the light-grown plants diminished after the 22-h period while that measured in etiolated seedlings increased. The responsiveness of seedlings to BA was correlated with endogenous cytokinin levels in that enhancement of NR induction by exogenous BA was low in tissues which contained high concentrations of cytokinin at the time of BA application. These results may prove useful in interpretation of gene responses to exogenous plant growth regulators.     

beta]-Tubulin Accumulation and DNA Replication in Imbibing Tomato Seeds

The activation of the cell cycle in embryo root tips of imbibing tomato (Lycopersicon esculentum Mill. cv Lerica) seeds was studied by flow cytometric analyses of the nuclear DNA content and by immunodelection of [beta]-tubulin. With dry seeds, flow cytometric profiles indicated that the majority of the cells were arrested at the G1 phase of the cell cycle. In addition, [beta]-tubulin was not detectable on western blots. Upon imbibition of water, the number of cells in G2 started to increase after 24 h, and a 55-kD [beta]-tubulin signal was detected between 24 and 48 h. Two-dimensional immunoblots revealed at least three different [beta]-tubulin isotypes. Thus, [beta]-tubulin accumulation and DNA replication were induced during osmotic priming. These processes, as well as seed germination rate, were enhanced upon subsequent imbibition of water, compared with control seeds that imbibed but were not primed. By contrast, when aged seeds imbibed, DNA replication, [beta]-tubulin accumulation, and germination were delayed. In all cases studied, both DNA replication and [beta]-tubulin accumulation preceded visible germination. We suggest that activation of these cell-cycle-related processes is a prerequisite for tomato seed germination. Furthermore, [beta]-tubulin expression can be used as a parameter for following the initial processes that are activated during seed imbibition.     

Polyamine oxidase activity and polyamine content in maize during seed germination

Polyamine oxidase (PAO, EC 1.5.3.3) activity and polyamine content in the cell wall and soluble fractions obtained from embryos, endosperms and shoots and roots of etiolated or green seedlings of maize ( Zea mays L. cv. WF9) during the first 7 days of germination were investigated. Polyamine content was also determined in the trichloroacetic acid-soluble (free polyamines) and trichloroacetic acid insoluble (bound polyamines) fraction obtained from the same tissues. PAO activity, determined by the radiometric method based on the recovery of the labelled reaction product 1-pyrroline, was mostly localized in the cell wall fraction. The activity was very low in embryos and endosperms and present in traces in roots. In etiolated shoots PAO activity increased sharply, while in green shoots it was low and increased slowly. No polyamines were found in the cell wall fraction and only putrescine was detected in the soluble fraction, with the exception of the embryo, where spermidine and spermine were also present. In the TCA-soluble fraction of embryos, putrescine increased during imbibition, while spermidine and spermine decreased; in the endosperm no relevant changes in polyamines occurred. In the same fraction of green and etiolated seedlings, putrescine increased, giving a peak at days 3–5, while spermidine decreased to very low levels. The amount of bound polyamines was 1–4% of the free ones. The pattern of PAO activity seems to be unrelated to endogenous free polyamine content, which is the same in shoots and roots of etiolated and green seedlings. Enzyme activity, very low in ungerminated seeds, increased continuously during the progression of germination, especially in etiolated shoots, indicating a possible involvement in cell wall formation.     

Cell division and subsequent radicle protrusion in tomato seeds are inhibited by osmotic stress but DNA synthesis and formation of microtubular cytoskeleton are not

We studied cell cycle events in embryos of tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds during imbibition in water and during osmoconditioning ("priming") using both quantitative and cytological analysis of DNA synthesis and beta-tubulin accumulation. Most embryonic nuclei of dry, untreated control seeds were arrested in the G(1) phase of the cell cycle. This indicated the absence of DNA synthesis (the S-phase), as confirmed by the absence of bromodeoxyuridine incorporation. In addition, beta-tubulin was not detected on western blots and microtubules were not present. During imbibition in water, DNA synthesis was activated in the radicle tip and then spread toward the cotyledons, resulting in an increase in the number of nuclei in G(2). Concomitantly, beta-tubulin accumulated and was assembled into microtubular cytoskeleton networks. Both of these cell cycle events preceded cell expansion and division and subsequent growth of the radicle through the seed coat. The activation of DNA synthesis and the formation of microtubular cytoskeleton networks were also observed throughout the embryo when seeds were osmoconditioned. However, this pre-activation of the cell cycle appeared to become arrested in the G(2) phase since no mitosis was observed. The pre-activation of cell cycle events in osmoconditioned seeds appeared to be correlated with enhanced germination performance during re-imbibition in water.     

Protective effect of inhibitors of succinate dehydrogenase on wheat seedlings during osmotic stress

The effect of malonate and sedaxane, a compound with the fungicidal effect which act as succinate dehydrogenase inhibitors, on the resistance of etiolated wheat seedlings (Triticum aestivum L.) to osmotic stress caused by 12% PEG 6000 solution, was studied. The presowing treatment of seeds with 0.3 mM sedaxane solution significantly reduced the inhibitory effect of osmotic stress on seedling growth. The protective effect of 10 mM malonate was significant when it was added to the incubation medium of the roots; the effect of preseeding treatment with malonate was less significant. Unlike malonate, malate had no positive effect on seedling growth under osmotic stress. The activity of succinate dehydrogenase and the hydrogen peroxide content decreased in seedlings after the treatment of roots with malonate and sedaxane. Pretreatment with sedaxane and the addition of malonate to the incubation medium of roots prevented the accumulation of a lipid peroxidation product, malondialdehyde, which is caused by osmotic stress, and increased peroxidase activity. It was concluded that the stress-protective effect of sedaxane and malonate on wheat seedlings might be due to the inhibition of succinate dehydrogenase-dependent formation of reactive oxygen species and the prevention of oxidative cell damage.     

Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis

It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.     

Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment.

The principal objectives of the space experiment, BRIC-AUX on STS 95, were the integrated analysis of the growth and development of etiolated pea and maize seedlings in space and a study of the effects of microgravity conditions in space on auxin polar transport in these segments. Microgravity significantly affected the growth and development of etiolated pea and maize seedlings. Epicotyls of etiolated pea seedlings were the most oriented toward about 40 to 60 degrees from the vertical. Mesocotyls of etiolated maize seedlings were curved at random during space flight but coleoptiles were almost straight. Finally the growth inhibition of these seedlings in space was also observed. Roots of some pea seedlings grew toward to the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles, which were germinated and grown under microgravity conditions in space, were significantly low as compared with those grown on the ground of the earth. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.     

Cell Cycle Activity and β-Tubulin Accumulation During Dormancy Breaking of Acer platanoides L. seeds

Cell cycle events in embryo axes of Norway maple (Acer platanoides L.) seeds were studied during dormancy breaking by flow cytometric analyses of the nuclear DNA content and by immunodetection of β-tubulin. Most embryonic nuclei of dry, fully matured seeds were arrested in the G₂ phase of the cell cycle. In addition, the lowest content of β-tubulin was detected in dry, mature seeds. Imbibition in water and cold stratification resulted in a decrease in the number of nuclei in G₂, and a simultaneous increase in β-tubulin content. In germinated seeds the content of β-tubulin was the highest and the number of cells in G₂ was the lowest. Both cell cycle events preceded cell expansion and division and subsequent growth of the radicle through the seed coat. The anatomical investigation has proved that the main reason for decrease in the number of nuclei in G₂ is mitosis, started with seeds germination (radicle protrusion). The activation of the cell cycle and the β-tubulin accumulation were associated with embryo dormancy breaking. This revised version was published online in July 2006 with corrections to the Cover Date.     

Endosperm protein of wheat seed as a determinant of seedling growth

Seed of a Mexican semidwarf wheat (Triticum aestivum L. cv. Inia 66), was obtained from a nitrogen fertilizer field trial grown in Mexico. A high positive correlation was obtained between seed protein content and seedling dry weight after 3 weeks growth (r = +0.92^**). The seedling dry weight was positively related to the protein content of the aleurone layer and endosperm, but not to the embryo. Small, 35 milligrams, high protein seeds (4.7 milligrams protein per seed) produced larger seedlings than large, 45 milligrams, low protein seeds (4.3 milligram protein per seed). There was no difference in the weight or protein content of embryos from low and high protein seeds and their growth was similar. Composite seeds of the two protein levels were produced by transferring embryos from one endosperm type to the other. After 4 weeks, there was no difference between the different embryo types grown on the same endosperm type. High protein endosperm produced more vigorous seedlings regardless of the embryo type grown on it, indicating that the factor(s) responsible for the greater growth of high protein seed is in the endosperm.     

Germination ecophysiology of Annona crassiflora seeds

BACKGROUND AND AIMS: Little is known about environmental factors that break morphophysiological dormancy in seeds of the Annonaceae and the mechanisms involved. The aim of this study was to characterize the morphological and physiological components of dormancy of Annona crassiflora, a tree species native to the Cerrado of Brazil, in an ecophysiological context. METHODS: Morphological and biochemical characteristics of both embryo and endosperm were monitored during dormancy break and germination at field conditions. Seeds were buried in the field and exhumed monthly for 2 years. Germination, embryo length and endosperm digestion, with endo-beta-mannanase activity as a marker, were measured in exhumed seeds, and scanning electron microscopy was used to detect cell division. The effect of constant low and high temperatures and exogenous gibberellins on dormancy break and germination was also tested under laboratory conditions. KEY RESULTS: After burial in April, A. crassiflora seeds lost their physiological dormancy in the winter months with lowest monthly average minimum temperatures (May-August) prior to the first rainfall of the wet season. The loss of physiological dormancy enabled initiation of embryo growth within the seed during the first 2 months of the rainy season (September-October), resulting in a germination peak in November. Embryo growth occurred mainly through cell expansion but some dividing cells were also observed. Endosperm digestion started at the micropylar side around the embryo and diffused to the rest of the endosperm. Exogenous gibberellins induced both embryo growth and endo-beta-mannanase activity in dormant seeds. CONCLUSIONS: The physiological dormancy component is broken by low temperature and/or temperature fluctuations preceding the rainy season. Subsequent embryo growth and digestion of the endosperm are both likely to be controlled by gibberellins synthesized during the breaking of physiological dormancy. Radicle protrusion thus occurred at the beginning of the rainy season, thereby maximizing the opportunity for seedlings to emerge and establish.     

STS-95 space experiment for plant growth and development, and auxin polar transport.

The principal objective of the space experiment, BRIC-AUX on STS-95, was the integrated analysis of the growth and development of etiolated pea and maize seedlings in space, and the effect of microgravity conditions in space on auxin polar transport in the segments. Microgravity conditions in space strongly affected the growth and development of etiolated pea and maize seedlings. Etiolated pea and maize seedlings were leaned and curved during space flight, respectively. Finally the growth inhibition of these seedlings was also observed. Roots of some pea seedlings grew toward the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles which were germinated and grown under microgravity conditions in space were significantly low. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and extremely promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.     

Changes in the content of modified nucleotides of total transfer RNA of wheat seedlings during greening

The contents in minor nucleotides of total transfer RNA (tRNA) of etiolated and light-grown wheat (Triticum aestivum L.) seedlings and of seedlings illuminated for 24 or 48 h were examined. The total tRNA of seedlings illuminated 24 h contained more, and that from seedlings illuminated 48 h still more modified nucleotides than that from etiolated ones. Thus, the appearance of the characteristic minor nucleotides of tRNA of light-grown wheat seedlings needs a rather long greening period, of at least 48 h.     

外源氯化胆碱可提高小麦线粒体膜的流动性

分别用ANS、DPH及16 -NS三种不同的标记物标记小麦黄化苗的线粒体 ,研究氯化胆碱 (cholinechloride,CC)对线粒体膜的荧光光谱、平均微粘度 (η )及ESR图谱的影响。结果表明 ,0.21 -1.79mmol·L-1 的CC均能显著降低线粒体膜的荧光强度、η 值及ESR图谱的序参数 (S)和旋转相关时间 (τc) ,表明CC可增加线粒体膜的流动性。为揭示CC的提高植物抗冷机制提供依据     

Effect of the antioxidant ionol (BHT) on growth and development of etiolated wheat seedlings: control of apoptosis, cell division, organelle ultrastructure, and plastid differentiation

Ionol (BHT), a compound having antioxidant activity, at concentrations in the range 1-50 mg/liter (0.45·10^-5-2.27·10^-4 M), inhibits growth of etiolated wheat seedlings, changes the morphology of their organs, prolongs the coleoptile life span, and prevents the appearance of specific features of aging and apoptosis in plants. In particular, BHT prevents the age-dependent decrease in total DNA content, apoptotic internucleosomal fragmentation of nuclear DNA, appearance in the cell vac-uole of specific vesicles with active mitochondria intensively producing mtDNA, and formation of heavy mitochondrial DNA ( = 1.718 g/cm³) in coleoptiles of etiolated wheat seedlings. BHT induces large structural changes in the organization of all cellular organelles (nucleus, mitochondria, plastids, Golgi apparatus, endocytoplasmic reticulum) and the formation of new unusual membrane structures in the cytoplasm. BHT distorts the division of nuclei and cells, and this results in the appearance of multi-bladed polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces intensive synthesis of pigments, presumably carotenoids, and the differentiation of plastids with formation of chloro- or chromoplasts. The observed multiple effects of BHT are due to its antioxidative properties (the structural BHT analog 3,5-di-tert-butyltoluene is physiologically inert; it has no effect similar to that of BHT). Therefore, the reactive oxygen species (ROS) controlled by BHT seem to trigger apoptosis and the structural reorganization of the cytoplasm in the apoptotic cell with formation of specific vac-uolar vesicles that contain active mitochondria intensively producing mtDNA. Thus, the inactivation of ROS by BHT may be responsible for the observed changes in the structure of all the mentioned cellular organelles. This corresponds to the idea that ROS control apoptosis and mitosis including formation of cell wall, and they are powerful secondary messengers that regulate dif-ferentiation of plastids and the Golgi apparatus in plants.     

Regional growth patterns in the hypocotyls of etiolated and green cress seedlings in light and darkness

Abstract. A system is described whereby seedling development can be analysed in terms of growth rates of specific 1 mm regions of the hypocotyl. The technique involves time-lapse photography of marked hypocotyls in a specially designed chamber which accommodates seedlings in various orientations with respect to gravity, and under irradiation regimes differing in light quality, quantity and direction. The results of a preliminary study of the upward growth of etiolated or green cress seedlings in darkness or overhead while light are reported. Highest growth rates in etiolated seedlings were observed in zones in the upper one-third of ihe hypocotyl. In green seedlings, growth was more prominent within the subapical zones. Light further restricted growth of the median and basal zones in both types of seedling. However, in their immediate responses to the onset of irradiation, green and etiolated seedlings differed markedly. In etiolated seedlings, recovery of growth at the apex was accompanied by the development of inhibition in the median-basal regions; green seedlings showed a transient inhibition of growth in the apical zone together with a strong immediate inhibition in the median-basal regions.     

Automorphosis of etiolated pea seedlings in space is simulated by a three-dimensional clinostat and the application of inhibitors of auxin polar transport

Etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown under microgravity conditions in space show automorphosis: bending of epicotyls, inhibition of hook formation and changes in root growth direction. In order to determine the mechanisms of microgravity conditions that induce automorphosis, we used a three-dimensional clinostat and obtained the successful induction of automorphosis-like growth of etiolated pea seedlings. Kinetic studies revealed that epicotyls bent at their basal region towards the clockwise direction far from the cotyledons from the vertical line (0 degrees) at approximately 40 degrees in seedlings grown both at 1 g and in the clinostat within 48 h after watering. Thereafter, epicotyls retained this orientation during growth in the clinostat, whereas those at 1 g changed their growth direction against the gravity vector and exhibited a negative gravitropic response. On the other hand, the plumular hook that had already formed in the embryo axis tended to open continuously by growth at the inner basal portion of the elbow; thus, the plumular hook angle initially increased; this was followed by equal growth on the convex and concave sides at 1 g, resulting in normal hook formation; in contrast, hook formation was inhibited on the clinostat. The automorphosis-like growth and development of etiolated pea seedlings was induced by auxin polar transport inhibitors (9-hydroxyfluorene-9-carboxylic acid, N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid), but not by anti-auxin (p-chlorophenoxyisobutyric acid) at 1 g. An ethylene biosynthesis inhibitor, 1-aminooxyacetic acid, inhibited hook formation at 1 g, and ethylene production of etiolated seedlings was suppressed on the clinostat. Clinorotation on the clinostat strongly reduced the activity of auxin polar transport of epicotyls in etiolated pea seedlings, similar to that observed in space experiments (Ueda J, Miyamoto K, Yuda T, Hoshino T, Fujii S, Mukai C, Kamigaichi S, Aizawa S, Yoshizaki I, Shimazu T, Fukui K (1999) Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment. J Plant Res 112: 487492). These results suggest that clinorotation on a three-dimensional clinostat is a valuable tool for simulating microgravity conditions, and that automorphosis of etiolated pea seedlings is induced by the inhibition of auxin polar transport and ethylene biosynthesis.