The Effect of Defoliation on Carbohydrate, Protein and Leghaemoglobin Content of White Clover Nodules

Single white clover plants grown in pots of Perlite in a controlledenvironment and completely dependent on N₂ fixation were defoliatedto various degrees (46–85 per cent of shoot weight removed).The soluble protein content of nodules declined by about 20per cent and leghaemoglobin content by 50 per cent in the first4–7 d after defoliation but increased again to controllevels as new leaf tissue appeared. In the short term (2–3h) carbohydrate content of nodules declined to different extentsdepending on the severity of defoliation. The initial declinein N₂ fixation and the respiration associated with it, appearednot to be related to the instantaneous carbohydrate contentof nodules but rather to the supply of current photosynthatefrom the shoot. After 24–48 h, however, the carbohydratecontent of nodules had declined to low levels, regardless ofthe severity (46 or 71 per cent shoot removed) of defoliation.As new leaf tissue appeared carbohydrate levels in all partsof the plant gradually recovered towards control levels. Microscopic examination of nodule sections indicated that onlyafter very severe defoliation (80–85 per cent shoot removed)was nodule deterioration evident. Even here, as the plant establishednew leaves, the damage to nodules was repaired and no noduleloss was apparent. Trifolium repens, white clover, defoliation, carbohydrate, protein, leghaemoglobin     

Diurnal Production and Utilization of Photosynthate in Nodulated White Clover

Gordon, A. J., Mitchell, D. F., Ryle, G. J. A. and Powell, C.E. 1987. Diurnal production and utilization of photosynthatein nodulated white clover.—J. exp. Bot. 38: 84–98. A steady-state ¹⁴C-labelling technique was used to examine thediurnal carbon fixation, storage and export characteristicsof white clover leaves. Approximately 70% of fixed carbon wasexported to other organs during the photoperiod. The remainingcarbon was stored mainly as starch (80% at the end of the photoperiod)with smaller amounts of sucrose, hexoses and charged compounds.Carbon export from the leaf at night was provided by remobilizationof starch. During the photoperiod it was estimated that c.60% of carbonexported from the leaf was directed towards the nodulated root;45% to nodules and 15% to roots. The 40% directed towards theshoot was supplemented by a further 11% of carbon (in the formof amides) re-exported from the nodules. During the photoperiod, all organs of the plant accumulatedcarbohydrate which was available for use during darkness, inconjunction with a diminished supply of exported carbon fromleaves. Nodules exhibited a striking pattern of carbohydratestorage and depletion. The levels of sucrose and starch in thenodules at the end of the photoperiod were sufficient to maintainN₂ fixation for 8–9 h of the 12 h dark period. We proposethat continued import from leaves provided the additional sucrosenecessary to support undiminished nodule function throughoutthe entire dark period. Key words: White clover, photosynthate, starch, carbohydrate, nodules, N₂ fixation     

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     

Axillary Bud Development in White Clover in Relation to Defoliation and Shading Treatments

A period of growth under shade netting in the glasshouse allowedthe cultivation of white clover stolons with an accumulationof undeveloped axillary buds similar to that often found onstolons from grass/clover swards. The subsequent capacity ofthese nodes to develop branches under different circumstanceswas investigated in three experiments. Removal of the laminaeand petioles subtending sets of four buds along a stolon reducedthe rate at which branches were initiated from the buds. Treatmentsin which petioles, or petioles plus laminae, were retained initiatedbranches more quickly. Shading the stolons reduced both therate of initiation and the percentage of buds which developed,unless both petioles and laminae were retained. There was someevidence that conditions applied to individual buds may actin the same way as the same conditions applied to sets of fourbuds and that illuminated nodes may depress the performanceof neighbouring shaded notes. Fewer buds developed at older nodes than at younger nodes duringthe summer, but during the autumn younger buds initially developedmore slowly than older buds. This suggests that buds can developat a younger nodal age in summer than in winter. When leafless stolons were cut up into component internodesbuds developed faster than on intact stolons, provided the budwas located at the end of the internode nearest the main stolongrowing point. If the bud was at the other end, branch developmentwas slower than on intact stolons. The results are discussedin relation to clover growth in sward conditions. White clover, Trifolium repens, axillary bud development, branching, growing points, defoliation, shading     

Short-term Changes in CO2 Evolution Associated with Nitrogenase Activity in White Clover in Response to Defoliation and Photosynthesis

Direct, continuous measurements of the CO₂ evolution of rootnodules, calibrated by direct measurements of rate of ethyleneproduction, were utilized to determine the short-term responseof nitrogenase activity to defoliation and photosynthesis inwhite clover. Defoliation (removal of all expanded leaflets) generally resultedin a fall in nodulated root respiration within 10 min; mostrespiration associated with nitrogenase activity ceased within1–2 h. Darkening of the shoot also reduced nodulated root respirationwithin 10 min, but the subsequent fall in respiration, althoughof the same magnitude, was slower. The re-illumination of shootslargely reversed these effects. The inhibition of photosynthesisby DCMU largely simulated the effects of darkening the shoots. It is concluded that, in these white clover plants of 100 mgto 2.0 g total weight, current photosynthate provides the primarysource of energy for N₂ fixation. The mobilization of reserveenergy substrate appeared to play only a small role. The minimumtime interval of 10 min between onset of treatment and fallin nodule respiration probably reflects the time taken to exhaustthe assimilate in transit between leaf and nodule. Key words: White clover, N₂ fixation, Defoliation, Photosynthesis     

Effect of Carbohydrate Demand on the Remobilization of Starch in Stolons and Roots of White Clover (Trifolium repens L.) after Defoliation

White clover plants were grown from stolon tips in growth cabinetsand then defoliated. Thereafter, changes in the contents ofnon-structural carbohydrates such as starch, sucrose, glucose,fructose, maltose, and pinitol in stolons and roots were monitored.Initial contents of carbohydrate reserves, photosynthetic supplyof new carbohydrates and carbohydrate demand after defoliationwere varied by growing the plants at various CO₂ partial pressures,by varying the extent of defoliation and by removing eitherroots or stolon tips at the time of defoliation. Remobilization of carbohydrate reserves in stolons increasedproportionally to their initial contents and was greater whenplants had been severely defoliated, suggesting that carbohydrateswere remobilized according to availability and demand. Starchwas the predominant reserve carbohydrate. Starch degradationwas associated with decreased contents of sucrose, glucose andfructose in young stolon parts and roots but not in old stolonparts suggesting that starch degradation was not strictly controlledby the contents of these sugars. A decrease in the demand forcarbohydrates by removal of roots did not decrease starch degradationbut increased the contents of sucrose, glucose, and fructose.Removal of stolon tips decreased starch degradation and contentsof sucrose, glucose, and fructose. The results suggest thatstarch degradation was controlled by a factor other than sucrose,glucose, and fructose which was exported from stolon tips, e.g.gibberellin. Key words: White clover, storage carbohydrates, remobilization, regrowth     

Defoliation in White Clover: Regrowth, Photosynthesis and N2 Fixation

Single plants of white clover, grown in a controlled environmentand dependent for nitrogen on fixation in their root nodules,were defoliated once by removing approximately half their shoottissue. Their regrowth was compared with the growth of comparableundefoliated plants. Two similar experiments were carried out:in the first, plants were defoliated at 2.5 g, and in the secondat 1.2 g total plant d. wt. Defoliation reduced rate of N₂ fixation by > 70 per cent,rate of photosynthesis by 83–96 per cent, and rate ofplant respiration by 30–40 per cent. Nodule weights initiallydeclined following defoliation as a result of loss of carbohydratesand other unidentified components. No immediate shedding ofnodules was observed but nodules on the most severely defoliatedplants exhibited accelerated senescence. The original rates of N₂ fixation were re-attained after 5–6or 9 d regrowth, with increase in plant size at defoliation.In general, the rate of recovery of N₂ fixation was relatedto the re-establishment and increase of the plant's photosyntheticcapacity. Throughout the growth of both defoliated and undefoliatedplants nodule respiration (metabolism) accounted for at least23 ± 2 per cent of gross photosynthesis. The unit ‘cost’of fixing N₂ in root nodules, in terms of photosynthate, appearedto be unaffected by defoliation, except perhaps for plants veryrecently defoliated. Similarly, the percentage nitrogen contentsof shoot, root and nodules of defoliated plants became adaptedwithin a few days to those characteristic of undefoliated plants. Trifolium repens, white clover, N₂ fixation, defoliation, photosynthesis, respiration     

Influence of Carbon Dioxide Concentration on Growth, Carbohydrate Content, Translocation and Photosynthesis of White Clover

White clover ramets were grown at various carbon dioxide concentrations(200, 350 and 1000 µl 1^–1), defoliated and regrownat the same concentrations. Morphological characteristics, dryweights and non-structural carbohydrate contents of plant organs,diurnal variation of sugar and starch content of leaves, translocationof assimilates and photosynthesis were determined. Carbon dioxide concentration influenced the dry weights, butnot the number and size of the plant organs. However, defoliationof plants at low carbon dioxide concentration resulted in decreasedleaf size and stolon length. Carbon dioxide concentration influencedthe content and diurnal variation of starch and sugar in theleaves. Starch was accumulated at medium carbon dioxide concentrationand sugar at a higher concentration when the storage capacityfor starch seemed to be exceeded. Starch was preferentiallyaccumulated in the first and sugar in the second half of thelight period. Translocation was decreased during the periodsof accumulation. Sugar accumulation in the leaves seemed tobe a consequence of the imbalance between sink and source, whereasstarch accumulation seemed to follow an in-built diurnal pattern.Accumulation of both starch and sugar during the photoperiodwas followed by degradation and export during the dark period.Decreased dark export occurred at low carbon dioxide concentrationwhen neither starch nor sugar was accumulated during the photoperiod. Carbon dioxide, white clover, Trifolium repens L., growth, carbohydrates, starch, sugar, translocation, photosynthesis     

The Effect of Defoliation on the Nitrogen Economy of White Clover: Regrowth and the Remobilization of Plant Organic Nitrogen

Single plants of white clover (Trifolium repens) were establishedfrom stolon cuttings rooted in acid-washed silver sand. Allplants were inoculated with Rhizobium trifolii, and receivednutrient solution containing 0·5 mg ¹⁵N as either ammoniumor nitrate weekly for 12 weeks (i.e. 6 mg ¹⁵N in total). Plantswere then leniently defoliated or left intact, and the labelledN supply was replaced with unlabelled N. Lenient defoliationremoved fully expanded leaves only, leaving immature leaveswhich accounted for 50–55% of the total; growing pointnumbers were not reduced. Nodules, leaves and growing pointswere counted over the following 21 d period, and d. wts, N contents,and ¹⁵N enrichments of individual plant organs were determined. Defoliated plants had fewer nodules, but numbers of growingpoints were unaffected by defoliation. The rates of both leafemergence and expansion were accelerated in defoliated plants;in consequence the number of young leaves remained less thanin intact plants until day 21. Total dry matter (DM) and N accumulationwere less in defoliated plants, and a greater proportion oftotal plant DM was invested in roots. About 97 % of plant totalN was derived from fixed atmospheric N, but there was incompletemixing of fixed and mineral N within the plant. Relatively moremineral N was incorporated into roots, whereas there was relativelymore fixed N in nodules. There was isotopic evidence that Nwas remobilized from root and stolon tissue for leaf regrowthafter defoliation; approximately 2 % of plant N turned overdaily in the 7-d period after defoliation, and this contributedabout 50% of the N increment in leaf tissue. White clover, Trifolium repens L. cv. SI84, lenient defoliation, N economy, regrowth, N remobilization     

Assimilate Partitioning in Three White Clover Cultivars in the Autumn, and the Effect of Defoliation

Partitioning of recently assimilated¹⁴C in three cloned whiteclover cultivars (Aberherald, Grasslands Huia and Sandra), grownoutdoors, was determined at the end of Aug., the end of Sep.and the end of Oct. to examine the relative strength of differentsinks within the plant when growing conditions increasinglyfavour carbohydrate accumulation in relation to growth. Also,the effect of removing two out of three expanded leaves on¹⁴Cpartitioning was studied. Export of¹⁴C from leaves increasedfrom late Aug. to late Oct. More¹⁴C was partitioned to the rootsand less to the apex at the Sep. and Oct. harvests than at theharvest in Aug., irrespective of cultivar. Severe defoliationresulted in more¹⁴C being partitioned to the apex and less tothe main stolon irrespective of cultivar and harvest occasion.Sandra (a cultivar of northern origin) generally partitionedmore¹⁴C to the stolon and less to the apex than did the othertwo cultivars. Trifolium repens L.; white clover; assimilate partitioning; defoliation; photosynthesis; autumn growth     

The Effect of Lenient Defoliation on the Nitrogen Economy of White Clover: The Contribution of Mineral Nitrogen to Plant Nitrogen Accumulation During Regrowth

Single plants of white clover (Trifolium repens L.) were grownfrom stolon cuttings rooted in sand. All plants were inoculatedwith Rhizobium trifolii, and for 14 weeks received nutrientsolution containing 0.5 mg N each week, as either ammonium ornitrate. Plants were then leniently defoliated or were leftintact and a ¹⁵N-labelled N source was applied at intervalsof 4 d to replace the unlabelled N. Lement defoliation removedfully expanded leaves only; the remaining immature leaves accountedfor 39–44% of the total. At harvests over the following21 d, leaf numbers were counted and dry matter (DM), N contentsand ¹⁵N enrichments of individual plant organs were determined. Rates of leaf emergence and expansion were accelerated in defoliatedplants; numbers of young leaves were similar in defoliated andintact plants. Total DM and N content were less in defoliatedthan intact plants and were not affected by form of N supplied.DM of young leaves, growing points and stolons and N contentof young leaves were, however, greater when ammonium ratherthan nitrate N was supplied. Rates of increase in the contentof plant total N were 8.2 ± 1.36 mg N d^-1 and 10.2±1.82 mg N d^-1 in defoliated and intact plants respectively.The increases were predominantly due to N₂ fixation, since recoveryof ¹⁵N showed that less than 1% of the increment in plant totalN was assimilated mineral N. Nevertheless, the contributionof mineral N to plant total N was 50% more in defoliated thanin intact plants; higher amounts of mineral N were found particularlyin young leaves and growing points. Partitioning of mineralN to nodulated roots increased over time and was greater whenammonium rather than nitrate N was present. White clover, Trifolium repens L. cv. S184, lenient defoliation, N accumulation, N₂ fixation     

The Influence of Leaf Position and Defoliation on the Assimilation and Translocation of Carbon in White Clover (Trifolium repens L.) 1. Carbon Distribution Patterns

The assimilation of carbon (C) by, and distribution of ¹⁴C from,leaves at each end of an unbroken sequence of ten mature leaveson the main stolon of clonal plants of white clover (Trifoliumrepens L.) were measured to identify intra-plant factors determiningthe direction of C movement from leaves. Leaves at two intermediatepositions were also measured. Localized movement of ¹⁴C to sinks at the same node as, or atthe one to two nodes immediately behind, the fed leaf accountedfor 40–50% of the total ¹⁴C exported by all measured leaves.A further 50–60% of exported ¹⁴C was therefore availablefor more-distant sinks, and the direction of translocation ofthis C was determined by the relative total strength or demand(number x size x rate of activity or growth) of sinks forwardof, or behind, the leaf in question. Thus 85% of the ¹⁴C exportedfrom the youngest measured leaf moved toward the base of thestolon, while about 60% of the ¹⁴C exported from the oldestleaf moved acropetally. Defoliating plants to leave just one mature leaf on the mainstolon (at any one of the same four positions studied in undefoliatedplants), and no leaves on branches, resulted in: (1) increasednet photosynthetic rate in all residual leaves: (2) increased%export of fixed C from one of the four leaves; (3) increasedexport to the main stolon apex from all except the eldest leaf;(4) increased export to branches from three of the four leaves;and (5) decreased export to stolon tissue and roots from allleaves, within 3 d of defoliation. These responses would seemto ensure the fastest possible replacement of lost leaf areaand, thus, restoration of homeostatic growth. The observed patternsof C assimilation and distribution in both undefoliated anddefoliated white clover plants are consistent with the generalrules of source-sink theory; the distance between sources andcompeting sinks, and relative sink strength, emerge as the mostimportant intra-plant factors governing C movement. These resultsemphasize the need to consider plant morphology, and the modularnature of plant growth, when interpreting patterns of resourceallocation in clonal plants, or plant responses to stressessuch as partial defoliation. Trifolium repens L, white clover, photosynthesis, assimilate translocation, defoliation     

Regrowth by Swards of Subterranean Clover after Defoliation. 1. Growth, Non-structural Carbohydrate and Nitrogen Content

Swards of subterranean clover (Trifolium subterraneum L.) atLAl 6 grown in N-free nutrient solution were subjected to threedefoliation treatments which removed 30, 70 and 80% of shootdry weight. Subsequent regrowth and changes in the concentrationsof carbohydrate and nitrogen in plant components were measuredat 0, 1, 5, 9 and 13 d after defoliation and compared with thosein uncut swards. The rate of shoot regrowth declined with increasing severilyof defoliation. In all defoliation treatments, growth was confinedto leaves for up to 5 d. Root growth ceased in all treatmentsfor a longer period. Reestablishment of the leaf area in severely-defoliatedswards was facilitated by the rapid opening of developing leavesand by changes in the allocation of carbon which favoured leafover branch and root, and lamina over petiole growth. Loss of carbohydrate and nitrogen from roots and branches lasting5–9 d was observed in the more severe defoliation treatments.Loss of protein (N x 6.25) exceeded that of total non-structuralcarbohydrate, and could have accounted for the nitrogen contentof new leaf during this period. Branches lost 62% of their initialcarbohydrate content compared with 25% from roots in the 80%cut swards. In contrast, roots, by virtue of their greater mass,were the principle source of mobilized nitrogen. Nitrogen accumulationceased in 80% cut swards for 9 d. However, carbohydrate levelsin the crown nodules were not severely depleted. It was concluded that partitioning of growth to lamina and themobilization of carbohydrates and nitrogen were important forrecovery from defoliation. Carbohydrates, carbon partitioning, defoliation, nitrogen, mobilization, regrowth, subterranean clover, Trifolium subterraneum L     

Effects of Acetylene and External Oxygen Concentration on the Respiratory Quotient (RQ) of Nodulated Roots of Soyabean and White Clover

Carbon dioxide output and oxygen uptake by intact nodulatedroots of soyabean and white clover were determined over a rangeof external oxygen concentrations (10 to 60%) in the presenceand absence of 10% acetylene. The responses of both sets ofroot systems were in agreement with the known characteristicsof the variable oxygen diffusion resistance of the nodules.These measurements were used to calculate the respiratory quotient(RQ, CO2/O2) for both total root respiration and nitrogenase-linkedrespiration values for each external oxygen concentration. Inthe absence of acetylene, the RQ values of soyabean total rootrespiration showed no significant response to external oxygen,with a mean of 1.06, but differences in values for white cloverroots were significant at P=0.05, with a mean of 1.09. The sametrend was shown for RQ values of nitrogenase-linked respiration,with mean values of 1.10 and 1.12 for soyabean and clover, respectively.The addition of acetylene caused a small but significant initialreduction in RQ of total root respiration with both soyabeanand white clover, as did the subsequent reduction of externaloxygen to 10%. For white clover, increases in external oxygenconcentration to 50% and 60% also produced significant increasesin the RQ of total root respiration compared to that in air.These variations were more pronounced for RQ values of nitrogenaselinkedrespiration. Results are discussed in relation to the use ofRQ values of unity in calculations of nodular oxygen diffusionresistance and the involvement of physical and biochemical processesin the regulation of oxygen supply to bacteroids. Key words: Respiratory quotient, nodules, oxygen, diffusion resistance     

H_2S对干旱胁迫下白三叶幼苗抗氧化防御能力的影响

以‘拉丁诺’白三叶(Trifolium repens cv.‘Ladino’)为试验材料,研究外源H2S处理对PEG6 000(聚乙二醇)模拟干旱胁迫下白三叶叶片相对含水量(RWC)、膜脂过氧化、活性氧成分、抗氧化酶、抗坏血酸-谷胱甘肽循环代谢和非酶抗氧化物质的影响,以揭示H_2S调控白三叶抗旱性的生理机制。结果显示:(1)0.2 mmol/L的外源NaHS(H_2S供体)能显著提高干旱胁迫下白三叶的叶片相对含水量,维持显著较低的电解质渗透率(EL)和丙二醛(MDA)含量。(2)与直接干旱胁迫相比,干旱胁迫下外源添加NaHS处理的白三叶叶片内超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性显著增强,抗坏血酸-谷胱甘肽循环代谢中关键酶抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、单脱水抗坏血酸还原酶(MDHAR)和谷胱甘肽还原酶(GR)活性及其抗氧化中间产物抗坏血酸(AsA)、谷胱甘肽(GSH)含量也显著提高。(3)叶片类黄酮、总酚和原花青素的含量在一定的胁迫时间范围内亦显著增加,并伴随着活性氧成分O_2~(-·)产生速率和H_2O_2水平降低。研究认为,外源H2S能通过促进干旱胁迫下白三叶体内的多重抗氧化防御能力来提高其幼苗的抗旱性。     

Influence of White Clover Mosaic Potexvirus Infection on the Endogenous Cytokinin Content of Bean

The cytokinin content in the primary leaves of bean (Phaseolus vulgaris) was monitored for 10 d after inoculation with white clover mosaic potexvirus. The cytokinins were isolated, purified, separated by high-performance liquid chromatography, and quantified by radioimmunoassay. The cytokinins detected at the time of inoculation (d 0) were: (a) the free bases, zeatin (Z), dihydrozeatin (DZ), and isopentenyladenine; (b) the riboside, DZ riboside (DZR); (c) the O-glucosides of DZ, DZR, and Z riboside; (d) the nucleotides, Z riboside-5′-monophosphate and isopentenyladenosine-5′-monophosphate; and (e) trace amounts of Z-9-glucoside and DZ-9-glucoside. During the 10 d after inoculation with white clover mosaic potexvirus, marked quantitative changes in this cytokinin profile were observed. The concentration of the free bases and DZR decreased, accompanied by an increase in the 9-glucosides and the nucleotides. Virus titer increased rapidly 3 d after inoculation, attaining a maximum level at d 5. This increase coincided with the increases in the 9-glucosides and the nucleotides. We propose that the decline in the cytokinin free bases and riboside may allow the increase of virus titer in bean and lead to the senescence of infected leaves.     

Oxygen Supply Limits Nitrogenase Activity of Clover Nodules After Defoliation

The aim of this study was to test the effect of oxygen partialpressure as a possible limiting factor of nitrogen fixationfollowing defoliation. The response of nitrogenase activity(C₂H₂-reduction) of defoliated and undefoliated white and redclover plants (Trifolium repens L. and Trifolium pratense L.)to either 19 kPa oxygen or 55 kPa oxygen was investigated. Priorto defoliation, white clover plants were grown for five weeksin a growth chamber, and red clover plants for 7 or 11 weeksin a glasshouse. The results included measurements of ¹⁶N₂-uptake. Increasing oxygen partial pressure from 19 to 55 kPa severelyrestricted nitrogenase activity of undefoliated white cloverplants; however, 2 h after complete defoliation, the same treatmentcaused a significant increase. A fivefold increase in nitrogenaseactivity upon exposure to the elevated oxygen partial pressurewas found at the end of a 24 h period. This beneficial effectdecreased gradually from 1 to 5 d after defoliation. The responseof recently defoliated red clover plants to 55 kPa oxygen partialpressure was similar to that of white clover, independentlyof plant age. The gradual recovery of nitrogenase activity duringthree weeks of regrowth was associated with a simultaneous changein the response to increased oxygen partial pressure, leadingagain to the response of undefoliated plants. These data suggested that lack of oxygen at the site of nitrogenfixation, resulting from a dramatic increase in oxygen-diffusionresistance, is the main factor limiting nitrogenase activityfollowing defoliation. Trifolium repens L., Trifolium pratense L., white clover, red clover, defoliation, regrowth, nodules, nitrogen fixation, nitrogenase activity, oxygen limitation