Effect of Temperature on Nitrogenase Activity in White Clover

Single, clonal plants of white clover were grown without inorganicnitrogen in four contrasting day/night temperature regimes,with a 12 h photoperiod, in controlled environments. Root andnodule respiration and acetylene reduction activity were measuredin a flow-through system during both day and night for plantsacclimated to day/night regimes of 23/18, 15/10 and 10/5 ?C.Similar measurements were made on plants acclimated to 20/15?C and stepwise at temperatures from 4 to 33 ?C. Peak rate of ethylene production, nitrogenase-linked respirationand basal root + nodule respiration increased approximatelylinearly from 5 to 23 ?C both in temperature-acclimated plantsand in plants exposed to varying measurement temperatures. Themeasured attributes did not vary significantly between day andnight. Temperatures above 23–25 ?C did not further enhancethe rate of ethylene production, which remained essentiallythe same up to the maximum measured temperature of 33 ?C. The measurements of nitrogenase-linked respiration between 5and 23 ?C, during both day and night, demonstrated a constant‘energetic cost’ of acetylene reduction of 2.9 µmolCO₂ µmol C₂H₄^–1,. Over the same temperature range,the approximate activation energy of acetylene reduction was60 kJ mol^–1. The integrated day plus night nitrogenase-linkedrespiration accounted for 13.4–16% of the plant‘snet shoot photosynthesis in a single diurnal period: there wasno significant effect of temperature between 5 and 23 ?C. Key words: Trifolium repens, white clover, temperature, N₂ fixation, respiration     

Pea Lectin Expressed Transgenically in Oilseed Rape Reduces Growth Rate of Pollen Beetle Larvae

In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T₀-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T₂-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T₂-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.     

The Effects of Thiamin and Nicotinic Acid on Meristematic Activity in Pea Roots

The results of experiments with isolated pea roots are presentedwhich show the effects on growth of providing nicotinic acidand thiamin in the medium. Certain of the experiments involvetwo experimental variables, the presence and absence of vitaminsin the medium and tip inocula of different lengths. Growth isanalysed in terms of overall length increase, increment in numberof cells, rates of division, and mature cell volume. It is shownthat both vitamins affect division, and in the case of vitaminB₁, through a primary effect which may be either on mature ormeristematic tissue. Evidence has not been obtained that withthe material of this investigation the vitamins have any effecton the growth of the cell. Further evidence is provided in favourof the hypothesis that division in the meristem depends on productsformed in the meristem, and on a different set of metabolitessynthesized in mature tissue.     

白三叶生物技术研究进展

白三叶(Trifolium repens L.)是品质优良、易于栽培的一种重要豆科牧草.随着分子生物学和植物基因工程的发展,在分子水平上对白三叶进行遗传改良的研究已经取得了部分成果.综述了近20年来生物技术在白三叶研究领域的应用.     

Development of Ascorbate Oxidase Activity and Its Iso-enzyme Pattern in the Roots of Pea Seedlings

Early in the germination of peas, ascorbate Oxidase activity increased markedly in the root and epicotyl, but not in the cotyledons. The activity in the root was suppressed by the administration of 6-methylpurine, thiouracil, cyclohexi-mide, thioproline, and phenylthiourea. Although enzyme development was inhibited by phenylthiourea and thioproline, root elongation was not affected by these reagents. As germination progressed, the ascorbate oxidase activity in the extract of the seedling's root could be separated into at least 3 to 7 isoenzymes.     

豌豆外源凝集素基因的克隆及序列分析

从豌豆幼叶分离基因组ＤＮＡ,设计特异引物,用聚合酶链式反应方法扩增出豌豆外源凝集素基因并克隆到Ｅ．ｃｏｌｉ质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＥｃｏＲＶ位点。进一步亚克隆至ｐＵＣ１９。序列分析表明,克隆到的片段大小为８３２ｂｐ,包含了豌豆外源凝集素基因完整的编码序列。该基因无内含子,同报道的已知序列相比,其核苷酸序列及推测的氨基酸序列的同源率分别为９９．６％和９８．９％。     

Effect of Phosphorus Deficiency on Phosphatase Activity of Cell Walls from Roots of Subterranean Clover

Clover (Trifolium subterraneum L. cv. Mt. Barker) was grownin solution culture with adequate (+P) or no phosphate (–P).Cell walls were extracted from roots in such a way that theywere uncontaminated by other cellular materials. Phosphataseactivity was assayed using p-nitro-phenylphosphate (NPP). Phosphatasebound to cell walls had a pH optimum between 5.0 and 6.0, irrespectiveof the P supply to the plants. Activity of phosphatase boundto cell walls increased with electrolyte concentration of theassay medium at pH 6.5 but not at pH 5.5. This increase in activitywas probably due to a higher degree of ionization of the cellwall at pH 6.5 than at pH 5.5, and to effects of high ionicstrength in decreasing the mutual repulsion of negatively chargedNPP from negative charges on the cell walls. Cell wall-boundphosphatase did not exhibit Michaelis-Menten kinetics: the concentrationof NPP at which activity was half the maximum rate (S_0.5) was0.7 mM for cell walls extracted from roots of both +P and –Pplants. Up to 30% of the phosphatase activity bound to cellwalls could be removed using buffer solutions of high pH andhigh ionic strength which contained Triton X100. Both soluble and cell wall-bound phosphatase(s) of roots increasedin activity with P deficiency. The phosphatase activity of cellwalls increased 1.5 fold as the P concentration in the rootsfell from 0.4–0.2% dry weight. Experiments with sterileroots of clover showed that increases in cell wall-bound phosphataseactivity associated with P deficiency were not due to microbialcontamination. It is argued that phosphatase(s) in cell wallsof roots could make a substantial contribution to the P nutritionof clover in soils deficient in inorganic phosphate by hydrolysingorganic phosphate compounds in the soil. Key words: Phosphatase, Clover, Roots, Phosphorus deficiency, Cell walls     

Phosphate Uptake and Transport by Roots and Stolons of Intact White Clover (Trifolium repens L.)

The extent to which phosphate can be absorbed directly fromthe outer medium by stolon internodes and contribute to thetotal accumulation of phosphate by intact plants of white clover(Trifolium repens L. cv. Blanca) was assessed in hydroponicexperiments in a controlled environment room. The uptake ofphosphate by intact roots or stolons was measured by sealinga segment (6-0 mm long) across a flow-cell in which ³²P-labellednutrient solution was circulated for 24 h, the rest of the rootsystem receiving unlabelled nutrient solution. The rate of uptakeof phosphate (µmol g^–1 d^–1 dry wt. basis)by roots was more than 300 times that by intact stolons. Pretreatmentof stolons by gentle abrasion to remove cuticle, so as to simulatethe condition of stolons in the field, increased the uptakeof phosphate 7-fold compared with that of intact stolons. However,the potential of stolons to contribute to the P status of whitedover in the field was calculated to be small (5%). When an incision was made through the hypodermal layer of stolons,the rate of phosphate uptake greatly increased, attaining 71%of that by root segments. This increase, which was greater athigher phosphate concentrations, indicates that the suberi.zedhypodermis constitutes a major barrier to the influx of phosphatein the stolon. After withholding phosphate for different time intervals, thesubsequent rate of phosphate uptake by roots was increased 2-3-foldafter 2 d phosphate deprivation and 3-4-fold after 6 d or 13d phosphate deprivation. A higher proportion of absorbed phosphatewas transported to shoots in phosphate-deprived plants. After1 d of uptake following restoration of the phosphate supply,the concentrations of labelled phosphate in shoots were greaterthan in control plants, although the concentrations of labelin roots was less. However, the rate of uptake of phosphateby stolons, following deprivation, was not significantly increased.These results suggest that the mechanism regulating the enhancedrate of phosphate loading into the xylem, initiated by a periodof phosphate deprivation, is specific to roots and is not inducedin stolons. The results are discussed in relation to the growth and acquisitionof phosphate by white clover in the field. Key words: Nutrient deficiency, phosphate, stolons, transport (ions), Trifolium repens     

Polyacrylamide Gel Electrophoresis (PAGE) and Densitometric Measurement of Enzyme Activity of the Pea Root Pathogen Aphanomyces euteiches in Pea Roots

A new method to measure enzyme activity of the fungal root pathogen Aphanomyces euteiches in pea roots is described. The specific enzymes of the fungus and the host were separated by polyacrylamide gel electrophoresis (PAGE) and the activity of fungal Glucose-6-phosphate dehydrogenase and Phosphoglucomutase were quantified by densitometry. Fungal activity could be correlated to the percentage infected root length and to the disease symptoms of the plants. The activity of A. euteiches was studied in a time course experiment with increasing levels of zoospore inoculum. The results indicated that an increase in inoculum level resulted in a faster disease development in the plants. The relation between fungal enzyme activity and infection level is discussed.     

Hairy Root-activation Tagging: a High-throughput System for Activation Tagging in Transformed Hairy Roots

Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system for activation tagging of genes in “transformed hairy roots”. The binary vector pHR-AT (Hairy Root-Activation Tagging) and its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest.     

Non-light-dependent Shikimate Pathway in Plastids from Pea Roots

Non-green plastids (leucoplasts) isolated from pea roots are shown to be considerably active in forming aromatic amino acids by the shikimate pathway which, in contrast to the chloroplast pathway, is independent of light. Supply of phosphoenolpyruvate and 3-dehydroquinate, 3-dehydroshikimate, shikimate and quinate effectively enhances the formation of aromatic amino acids suggesting an intra- or/and intercellular intermediate transport.     

Isolation of Casparian Strips from Pea Roots

Casparian strips were isolated from roots of pea seedlings.The plasma membrane was firmly attached to the wall of the individualcells in the isolated Casparian strip. The outer electrondenselamella of the plasma membrane, which is characteristic of themembrane of the Casparian strip, was not removed from the isolatedCasparian strip by treatment with either 1 M NaCl or 2 M sodiumthiocyanate. Although treatment with 1% Triton X-100 disruptedthe lamellar structure of the membrane and removed most of theelectron-dense material of the outer lamella, no detectablepolypeptides were extracted by treatment of isolated Casparianstrips with 1% Triton X-100. Small amounts of electron-densematerial remained after treatment with Triton X-100 and thismaterial seemed to be removed almost entirely by subsequenttreatment of the same sample with 2% SDS. Since polypeptidesof 46, 30 and 20 kDa, which were detected in the extract obtainedby treatment with SDS, were not detected in an extract of totalcell walls of pea roots, it seems possible that these polypeptidesare specific to the Casparian strip and, therefore, that theymay be responsible singly or together for the adhesion of theplasma membrane to the cell wall at the Casparian strip. (Received January 30, 1992; Accepted April 23, 1992)     

Effects of Nematicides and Plant Resistance on White Clover Performance and Seasonal Populations of Nematodes Parasitizing White Clover in Grazed Pasture

Root-infecting nematodes are a major cause of white clover, Trifolium repens, not reaching its potential in New Zealand pastures. Resistance and/or tolerance are the preferred control options. Greenhouse-based, recurrent selection programs have developed resistance to Meloidogyne trifoliophila and Heterodera trifolii, and a field-based program has developed tolerance. Lines from these programs were compared with commercial cultivars as controls in a series of field trials at four sites over 4 years. Resistant lines from the CCN program performed better than susceptible lines and as well as most cultivars, reflecting the high level of resistance developed in this greenhouse-based program. In stained root from Cambridge, numbers of CCN were lower in resistant lines than in cultivars; numbers in susceptible lines were intermediate. CCN resistance was also reflected to a lesser extent in the number of cysts counted in soil under resistant lines in Palmerston North. The root-knot nematode-resistant material performed better than the susceptible and as well as most cultivars. In one trial of CRKN-resistant lines, resistant and susceptible lines had similar numbers of CRKN which were both lower than the numbers in the cultivars; in the second trial, there were fewer CRKN in resistant than in susceptible lines or cultivars. The tolerant selections, developed under field conditions, performed as well as or better than the cultivars. The selections from the breeding programmes have exhibited strong agronomic potential across locations and years, and the best material has been crossed; progeny are being assessed in current field trials.     

Chlorophyll Formation in Excised Roots of Cucumber and Pea

The effects of red and blue light on the formation of chlorophyll in excised roots of cucumber and pea was investigated and compared with previously reported experiments on wheat roots. All three kinds of roots are similar in that in continuous red light no or very little chlorophyll is formed. and in blue light a considerable amount of chlorophyll is produced. Roots of cucumber and pea differ from those of wheat, in that red light is ineffective (or nearly so) even when combined with blue.     

青蒿素在露水草毛状根中的生物转化

露水草毛状根培养系中加入青蒿素培养８ｄ后,青蒿素转化去氧青蒿素。根据光谱数据,对去氧青蒿素的结构进行了鉴定。结果表明,通过水草毛状根能将青蒿素进行选择性还原为去氧青蒿素。     

The Influence of Frequent Defoliation and of Drought on Nitrogen and Sulphur in the Roots of Perennial Ryegrass and White Clover

Frequent defoliation and drought, imposed individually overa period of 60 days, both reduced substantially the root weightsof white clover grown in the field, while causing no reductionin the root weights of perennial ryegrass. Concentrations ofN and S in the root organic matter of the clover were reducedby between 14 and 25 per cent by both treatments, but with theryegrass concentrations were not reduced. Perennial ryegrass, white clover, roots, nitrogen, sulphur, defoliation, drought     

Azelaic Acid-Induced Enzymes of Phenolic Defense in Pea Roots

The treatment of pea roots with azelaic acid for 72 h led to a change in the content of 28 proteins: the content of 20 proteins decreased, and the content of 8 proteins (including the phenylpropanoid metabolism enzymes, which are involved in the synthesis of phytoalexins) increased.     

Influx and Efflux of Nitrate and Ammonium in Italian Ryegrass and White Clover Roots: Comparisons Between Effects of Darkness and Defoliation

Seedlings of Italian ryegrass (Lolium multiflorum Lam. cv. RVP)and clonal stolon cuttings of white clover (Trifolium repensL. cv. Blanca) were grown for 19 d in flowing solution culture,with N supplied as either 250 mmol m^–3 NO₃^– or NH₃⁺.Rates of net uptake, influx and translocation of NO₃^–and NH₄⁺ were then determined using ¹⁵N and ¹³N labelling techniques:between 3–5 h into the photoperiod following 8 h darknessfor white clover (CL), and for ryegrass plants that were eitherentire (IL) or with shoots excised 90 min prior to ¹³N influx(IC); and 75 min into the photoperiod following 37–39h darkness for ryegrass (ID). Rates of net uptake, influx andefflux of NH₄⁺ exceeded those of NO₃^– in IL and IC ryegrassplants: the opposite occurred in white clover (CL). The decreasein net uptake following defoliation of ryegrass was greaterfor NH₄⁺ (62%) than NO₃^– (40%). For NH₄⁺ this was associatedwith a large decrease in influx from 110 to 6.0µmol h^–1g^–1 root fr. wt; but for NO₃^–, influx only decreasedfrom 42 to 37 µmol h^–1 g^–1. Prolonged exposureto darkness (ID plants) also lowered net uptake of NO₃^–and NH₄⁺ by, respectively, 86% and 95% of IL levels. For NH₄⁺this was characterized by a large decrease in influx and a smalldecrease in efflux; whilst for NO₃^– the effect of a largedecrease in influx was reinforced by a smaller increase in efflux. The data were used to estimate the translocatory fluxes of NO₃^–(03–20µmol h^–1 g^–1) and NH₄⁺ (003–0.4µmolh^–1 g^–1), assimilation in the roots of NO₃^–(02–26µmol h^–1 g^–1) and NH⁺⁴ (05–89 µmolh^–1 g^–1), and the concentrations of NO₃^– (9–15mol m^–3) in the cytoplasmic compartment of the roots.The relevance of variable influx and efflux to models for theregulation of N uptake is discussed. Key words: Lolium multiflorum, Trifolium repens, influx, efflux, nitrate, ammonium, ¹³N