Effects of Benzyladenine on Bean Leaf Senescence and the Translocation of 14C-Assimilates

Treating primary leaves of bean plants with benzyladenine (BA) greatly increased the retention of photosynthetic assimilates in the treated leaves. Within 24 hours of treatment, the BA treated primary leaves retained 70 % of their assimilates and maintained this high level throughout the period studied. In contrast, the primary leaves of control plants retained 30 % at week 2, increased retention to 80 % between week 4 and 5 and dropped to 50 % during senescence at week 6. When the trifoliate leaves of 5 week old plants were fed ¹⁴CO₂, less than 1 % of the total activity was recovered from the BA treated leaves. It is concluded that the retardation of leaf senescence by BA on intact plants is not due to mobilization of metabolites from other plant parts, but is associated with a high retention of photosynthates.     

Leaf senescence and grain filling affected by post-anthesis high temperatures in two different wheat cultivars

High temperature is a major factor affecting grain yield and plant senescence in wheat growing regions of central and east China. In this study, two different wheat cultivars, Yangmai 9 with low-grain protein concentration and Xuzhou 26 with high-grain protein concentration, were exposed to different temperature regimes in growth chambers during grain filling. Four day/night temperature regimes of 34°C/22°C, 32°C/24°C, 26°C/14°C, and 24°C/16°C were established to obtain two daily temperatures of 28 and 20°C, and two diurnal day/night temperature differences of 12 and 8°C. Concentration of a lipid peroxidation product malondialdehyde (MDA), activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT), chlorophyll concentration (SPAD) in flag leaves and kernel weight were determined. Results show that activities of SOD and CAT in leaves increased markedly on 14 days after anthesis (DAA) for the high-temperature treatment (34°C/22°C) and then declined. As a result, MDA concentration in leaves increased significantly under high temperature (34°C/22°C and 32°C/24°C). Compared with optimum temperature treatment, high temperature reduced the concentration of soluble protein and SPAD values in flag leaves. Grain-filling rate increased slightly initially, but decreased significantly during late grain filling under high temperature. As a result, final grain weight was reduced markedly under high temperature. Decreases in the activities of SOD and CAT and increases in MDA concentration in leaves were more pronounced with a 12°C of day/night temperature difference when under high temperatures. Kernel weight was higher under 12°C of day/night temperature difference under optimum temperatures (24°C/16°C and 26°C/14°C). The responses to high-temperature regimes appeared to differ between the two wheat cultivars with different grain protein concentrations. It is concluded that a larger diurnal temperature difference hastened the senescence of flag leaves under high-temperature conditions, but retarded senescence under optimum temperature treatments of 26°C/14°C and 24°C/16°C.     

Benzyladenine-Directed Transport of Carbon-14 and Phosphorus-32 in Senescing Bean Plants

The distribution of radioactivity from applied sucrose.¹⁴C and³²P to various plant parts were studied in relation to the retardationof leaf senescence by applied benzyladenine (BA) in intact beanplants. In short-time experiments sucrose-¹⁴C was fed to theplants for 48 h through the second trifoliate leaf at weeklyintervals from the third to the eighth week after planting.In long-term experiments sucrose-¹⁴C was fed to all plants for48 h at the third week and changes in distribution examinedat weekly intervals up till the eight week. In both cases, BAapplied to the primary leaves of intact bean plants did notcause a directed mobilization of sucrose-¹⁴C. When the plantswere stripped, leaving the primary leaves and the terminal pod,and fed sucrose-¹⁴C or ³²P through the terminal leaflet of thesecond trifoliate leaf, the BA-treated leaf accumulated relativelymore radioactivity than the opposite water-treated leaf. Itwas concluded that the retardation of senescence by BA in theprimary leaves of intact bean plants does not result directlyfrom the mobilization of metabolites and nutrients from otherplant parts. It is therefore suggested that BA-induced longevityin the primary leaves of the intact plant is accomplished bymetabolic self-sustenance.     

Retardation of leaf senescence by benzyladenine in intact bean plants

Summary Benzyladenine (BA) applied to primary leaves of intact bean plants delayed the senescence of both the leaves and the entire shoot. The retardation of senescence was manifested in higher levels of chlorophyll, protein, RNA and ribonuclease activity at all stages of development. Also, the levels of incorporation of labelled precursors into protein and RNA were enhanced. The effect of BA was largely independent of light intensity and the compound did not act merely as a nitrogen source.     

The Importance of Roots in Regulating the Senescence of Soybean Primary Leaves

The role of roots in regulating primary leaf senescence of 14-day-old soybean seedlings was investigated. Compared with intact seedlings, the senescence of primary leaves is accelerated by removal of the root system but delayed if apical bud and the first trifoliate leaf are removed. No difference in senescence was found between intact seedlings and seedlings without roots, apical bud, and first trifoliate leaf. Lateral roots seem to play a predominant role in regulating primary leaf senescence. However, neither root nodules nor primary root play any function in senescence. Results indicate that benzyladenine (BA) at optimal concentration (2 mg/1) completely replaces the roots to prevent the senescence of primary leaves, whereas gibberellic acid (GA) and abscisic acid (ABA) accelerate. The effect of indole-3-acetic acid (IAA) to replace roots in preventing senescence depends on the season the young seedlings are grown. Additional, though indirect, information of acropetal transport of ABA is provided. In conclusion, it seems that cytokinins in lateral roots play a predominant role in leaf senescence and the normal supply of root cytokinins is important in leaf metabolism.     

Increases in Nuclear DNA Content without Mitosis in Benzyladenine-treated Primary Leaves of Intact and Decapitated Bean Plants

Primary leaves of intact bean plants (Phaseolus vulgaris L.cv. Yamashiro-kurosando-saito) were treated with benzyladenine(BA) beginning on the seventh day after sowing when cell proliferationin the leaves had finished. Nuclear DNA contents were measuredby cytofluorometry combined with 4',6-diamidino-2-phenylindole(DAPI) staining. In the untreated controls, most mesophyll andabaxial epidermal cells contained a nucleus whose DNA contentwas 2C; whereas most adaxial epidermal cells contained a 4Cnucleus. Benzyladenine treatment induced 4C nuclec in mesophylland abaxial epidermal cells; but BA induced 8C nuclei in adaxialepidermal cells. To compare the effects of endogenous cytokininaccumulation, bean plants were decapitated above the primaryleaves on day 7 and continually disbudded thereafter. Changesin the nuclear DNA content in primary leaves attached to thedecapitated plants was similar to that for BA-treated primaryleaves. No multinucleate cells were formed and no mitotic figureswere present in the BA-treated leaves or in the primary leavesof decapitated plants. Our results indicate that both BA treatmentand decapitation induced one round of nuclear DNA synthesiswithout mitosis in a large number of mesophyll and epidermalcells.     

Growth and Development in the Young Tomato: III. THE EFFECT OF NIGHT AND DAY TEMPERATURES ON VEGETATIVE GROWTH

Tomato seedlings were grown in a 12-hour day at constant andalternating day and night temperatures ranging from 10°to 30° C. The pattern of results was similar at light intensitiesof 400 and 800 f.c. The maximum rate of dryweight accumulationoccurred at a constant temperature close to 25° C. The effectsof day and night temperatures on total dry weight showed a considerabledegree of independence. The optimum day temperature was 25°C irrespective of the night temperature; the optimum night temperatureincreased from 18° to 25° C over the whole range ofday temperature. On average, day temperature affected totaldry weight twice as much as night temperature. High night temperaturesto some extent compensated for low day temperatures. The optimumday and night temperatures for leaf growth were both 25°C. On average day temperature affected leaf growth one and ahalf times as much as night temperature. By 12-hourly sampling it was shown that the cotyledons and leavesgrow throughout both day and night and that high night temperatureaccelerates nocturnal growth (cotyledons by cell expansion,young leaves by cell multiplication). Plants having receivedonly one night at 25° C, as compared with 15° C, showa slightly greater assimilation during the following light period,apparently as a consequence of increased photosynthetic surface.The respiratory loss in dry weight during darkness was not significantlyaffected by temperature over the range 15–25° C.     

Temperature effects on endogenous indole-3-acetic acid levels in leaves and stamens of the normal and male sterile 'stamenless-2' mutant of tomato (Lycopersicon esculentum Mill.)

The indole-3-acetic acid (IAA) concentration in leaves and stamens of the normal and a temperature-sensitive male sterile ‘stamenless-2′ (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.), grown under three temperature conditions, was measured by gas chromatography — mass spectrometry — selected ion monitoring (GC-MS-SIM) and by enzyme-linked immunosorbant assay (ELISA). At low (LTR, 18°C day/15°C night), intermediate (ITR, 23°C day/18°C night), and high temperatures (HTR, 28°C day/23°C night), the mutant leaves had approximately 10 to 20 times higher IAA concentrations, respectively, than the normal leaves under these temperature regimes. Similarly, the stamens of mutant flowers had approximately five and eight times higher IAA concentration at ITR and HTR, respectively, than the normal flowers. In the low temperature reverted mutant stamens, however, the level of IAA was similar to that in normal stamens. Also, with an increase in temperature, there was an increase in the level of IAA in the leaves and stamens of mutant plants. However, different temperatures had no appreciable effect on the IAA content of leaves and stamens of normal plants. It is suggested that the high IAA content in leaves and stamens of the stamenless-2 mutant is one of the factors associated with male sterility and carpellization of stamens in this mutant.     

Mode of high temperature injury to wheat. II. Comparisons of wheat and rice with and without inflorescences

High temperature injury to wheat ( Triticum aestivum L.) during grain development is manifested as acceleration of senescence. Experiments were conducted to elucidate the mode of senescence and site of high temperature responses. Wheat (cv. Chris) and rice ( Oryza sativa L. cv. Newbonnet), which have C₃ photosynthesis but different temperature responses, were grown with and without inflorescences under three temperature regimes after anthesis. Plant growth and constituents associated with senescence were measured weekly until physiological maturity. Increasing temperatures from 25°C/15°C to 35°C/25°C day/night after anthesis decreased growth, leaf viability, chlorophyll and protein concentrations, and RuBP carboxylase activity and increased protease and RNase activities in wheat. Inflorescence removal increased vegetative weights and slowed most senescence processes more in wheat than in rice, but did not alter the course of high temperature responses. Results are interpreted as indicating that diversion of nutrients from roots by inflorescence sinks at normal temperatures and by increased respiration at high temperatures caused similar responses. Source and sink activities appeared to be regulated jointly, probably by cytokinins from roots, during senescence at normal and elevated temperatures.     

Retardation of bean leaf senescence by benzyladenine and its influence on phosphate metabolism

The levels of glucose, sugar phosphates, and adenosine phosphates were determined in primary leaves of intact bean plants during normal senescence and compared to leaves in which senescence was delayed by application of benzyladenine (BA). In both cases there was a rise with time in the levels of glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate, and a decline in 2-phosphoglyceric acid, inorganic phosphate, and the adenosine phosphates (AMP, ADP, ATP). The levels of fructose 1,6-diphosphate remained fairly constant. Although the levels of hexose phosphates, adenosine phosphates, and inorganic phosphate were lower in the BA-treated leaves, the incorporation of ³²P into these compounds by 3- and 6-week-old plants was higher than in the controls. These results suggest that the retardation of leaf senescence by BA in intact bean plants is associated with increased utilization of metabolites, indicating a more rapid turnover of the adenosine phosphates. It is concluded that this effect is brought about by a regulatory coordination of metabolic processes in relation to energy production and utilization.     

Mode of high temperature injury to wheat during grain development

High temperature stress adversely affects wheat growth in many important production regions, but the mode of injury is unclear. Wheat ( Triticum aestivum L. cv. Newton) was grown under controlled conditions to determine the relative magnitude and sequences of responses of source and sink processes to high temperature stress during grain development. Regimes of 25°C day/15°C night, 30°C day/20°C night, and 35°C day/25°C night from 5 days after anthesis to maturity differentially affected source and sink processes. High temperatures accelerated the normal decline in viable leaf blade area and photosynthetic activities per unit leaf area. Electron transport, as measured by Hill reaction activity, declined earlier and faster than other photosynthetic processes at the optimum temperature of 25/15 °C and at elevated temperatures. Changes in RUBP carboxylase activities were similar in direction but smaller in magnitude than changes in photosynthesic rate. Increased protease activity during senscence was markedly accentuated by high temperature stress. Specific protease activity increased 4-fold at 25/15 °C and 28-fold at 35/25 °C from 0 to 21 days after initiation of temperature treatments. Grain-filling rate decreased from the lowest to the highest temperature, but the change was smaller than the decrease in grain-filling duration at the same temperatures. We concluded that a major effect of high temperature is acceleration of senescence, including cessation of vegetative and reproductive growth, deterioration of photosynthetic activities, and degradation of proteinaceous constituents.     

Influence of Soil and Air Temperature on I. Nucleotide Metabolism and Growth of Phaseolus vulgaris L. seedlings

Eight day old greenhouse grown Phaseolus vulgaris L. seedlingswere exposed to three different temperature regimes. The concentrationof the total free nucleotides, nucleoside triphosphates, RNAand protein was much more pronounced in seedlings exposed tothe interactive day/night temperature of 28 ?C (soil) and 15?C (air) of regime I. Seedlings treated to a low soil/air, day/nighttemperature of 15 ?C (regime II) had the lowest content of totalfree nucleotides, nucleoside triphosphates, RNA and protein.The nucleoside triphosphate content and energy charge valueof seedlings treated to a high soil/air, day/night temperatureof 28 ?C (regime III) declined much earlier than in the seedlingsexposed to temperature regimes I and II. The fresh and dry weightof seedlings exposed to temperature regime I was significantlyhigher than those seedlings exposed to the other two temperatureregimes. The slowest growth was observed in seedlings exposedto temperature regime II. The noticeably higher concentrationof ATP, pyridine nucleotides and root nodulation in seedlingsexposed to temperature regime I suggests that higher soil andlow air temperature enhances nitrogen metabolism in P. vulgarisseedlings.     

Retardation and Reversal of Senescence in Bean Leaves by Benzyladenine and Decapitation

During the early stages of growth when the primary leaves ofbeans (Phaseolus vulgaris L., cv. Kinghorn) are expanding, thereis an increase in chlorophylls, carotenoids and protein levelsas well as superoxide dismutase (SOD), catalase and peroxidaseactivities. However, following the initiation of flowering onday (d) 21 there is a steady decrease in all of these parametersexcept levels of peroxidase activity, and by d 35 the primaryleaves are also showing pronounced morphological symptoms ofsenescence. Following the onset of senescence, primary leaveswere also leaking and showed increased levels of malondialdehyde(MDA). In addition, there was an increase in the lipid phasetransition temperature of a membrane fraction consisting predominantlyof chloroplast membranes, which reflected the formation of gelphase lipid. By d 56, the leaves had abscinded. A single applicationof benzyladenine (BA) to the primary leaves or decapitationof the shoot above the primary leaf on d 9 retarded all of thesesenescence symptoms, with decapitation being more effectivethan BA. On d 35 when the primary leaves were pale green-yellow, a singleapplication of BA to the leaves or decapitation of the shootabove them reversed the symptoms of senescence. Decapitationwas more effective than BA as a means of reversal, causing chlorophyll,carotenoid and protein levels to rise to values higher thanthose present in the control leaves. Decapitation also reversedthe senescence-related changes in membrane phase properties,decreased membrane leakiness and reduced lipid peroxidation. (Received August 22, 1983; Accepted January 26, 1984)     

Effect of high temperature on active oxygen species,senescence and photosynthetic properties in cucumber leaves

To gain a physiological understanding of the effects of high temperatures on cucumber (Cucumis sativus L.), we subjected seedlings to heat treatment at daytime temperatures of 28 °C, 32 °C, and 36 °C for 7 h a day for 30 days. The amount of active oxygen species, indicators of senescence, and photosynthetic properties in the second and third leaves were determined at the start of temperature treatment and on the 15th and 30th days of treatment. The amount of active oxygen species superoxide in leaves was greatest in the high temperature zones on the 15th day of treatment, and the amount of hydrogen peroxide was greatest in the high temperature zones on both the 15th and 30th days of treatment. The reduction in the amount of protein and the increase in the amount of malondialdehyde, both indicators of senescence, were greatest in the high temperature zones on both the 15th and 30th days of treatment, and the amount of chlorophyll was lowest in the 36 °C zone on the 15th day, and lower in the high temperature zones on the 30th day. It is clear from these results that a large amount of active oxygen species is generated and accumulated in the leaves at high temperatures, and senescence is significantly accelerated. The photosynthetic properties of stomatal conductance, sub-stomatal CO₂ concentration, and transpiration rate were at the same level on both the 15th and 30th days of treatment in all three temperature treatment zones. No significant difference was seen in the net photosynthesis rate between the 28 °C and 32 °C zones, was lower in the 36 °C zone than the 32 °C zone on the 15th day, and lowest in the 36 °C zone on the 30th day. CO₂ intake and water absorption are only mildly affected by high temperatures, and the reduction in net photosynthesis rate due to the 36 °C high temperature stress suggests that the large amount of active oxygen species induces inhibition of photosynthesis and damage to the mechanism of photosynthesis.     

Accumulation of pathogenesis-related proteins in barley leaf intercellular spaces during leaf senescence

Accumulation of the pathogenesis-related (PR) proteins localised in intercellular spaces of barley primary leaves, chlorophyll content, structure of chloroplasts, and photosynthesis were examined during natural and in vitro induced leaf senescence (cultivation of whole plants in the dark or detached leaves under nutrient deficiency). Some of PR proteins accumulated during natural senescence, but their accumulation pattern was different from those of pathogen-induced as well as during in vitro-induced senescence, which indicate different molecular bases of these processes. Photosynthetic rate and chlorophyll content indicate that natural senescence of barley primary leaves began from 15th day after sowing. In 35-d-old first leaves, the chloroplasts showed typical characteristics of senescence as significant decrease of size, greater grana, and prominent plastoglobuli. The chloroplasts contained more grana under in vitro induced senescence and they had reduced length in the dark. Correspondingly, accumulation of PR proteins was detectable on about the 15th day but the content of some PR proteins increased in later stages of senescence. This revised version was published online in July 2006 with corrections to the Cover Date.     

THE EFFECT OF SHORT PERIODS OF HIGH TEMPERATURE DURING DAY AND NIGHT PERIODS ON PEA YIELDS

Karr , E. J. (Ohio State U., Columbus), A. J. Linck , and C. A. Swanson . The effect of short periods of high temperature during day and night periods on pea yields. Amer. Jour. Bot. 46(2) : 91-93. Illus. 1959.—The effect of high temperatures during periods of relatively short duration (3-4 days) at various stages following anthesis at the first bloom node was studied in relation to yield of peas at this node. Except for the periods of differential temperature treatments, the plants were maintained in a standard environment room (24°C., light, 12 hr.; 15°C., darkness, 12 hr.). Three different temperature regimes during the treatment periods were studied: high day temperature—standard night temperature (32°—15°C.) ; standard day temperature—high night temperature (24°—30°C.) ; and high day and night temperatures combined (32°—30°C.). The data reveal the existence of a relatively well-defined thermal-sensitive period, with maximal sensitivity to high day temperatures occurring at about 9-11 days from full bloom, and maximal sensitivity to high night temperatures occurring about 6-9 days from full bloom. High night temperatures proved more critical, resulting in a maximal reduction of 25% in yield, as opposed to about 8% for high day temperatures. The effect of high day and night temperatures combined tended to be roughly additive.     

Effects of Chilling Temperature on the Activity of Enzymes of Sucrose Synthesis and the Accumulation of Saccharides in Leaves of Three Sugarcane Cultivars Differing in Cold Sensitivity

The effects of short-term exposure to chilling temperature (10 °C) on sucrose synthesis in leaves of the cold-tolerant sugarcane cultivars Saccharum sinense R. cv. Yomitanzan and Saccharum sp. cv. NiF4, and the cold-sensitive cultivar S. officinarum L. cv. Badila were studied. Plants were grown at day/night temperatures of 30/25 °C, and then shifted to a constant day/night temperature of 10 °C. After 52-h exposure to the chilling temperature, sucrose content in the leaves of NiF4 and Yomitanzan showed a 2.5- to 3.5-fold increase relative to that of the control plants that had been left on day/night temperatures of 30/25 °C. No such increase was observed in Badila leaves. Similarly, starch content in the leaves of NiF4 and Yomitanzan was maintained high, but starch was depleted in Badila leaves after the 52-h exposure. During the chilling temperature, sucrose phosphate synthase (SPS; E.C.2.4.1.14) activity was relatively stable in the leaves of NiF4 and Yomitanzan, whereas in Badila leaves SPS activity significantly decreased. There was no significant change in cytosolic fructose-1,6-bisphosphatase activity for the three cultivars at the chilling temperature. This supports the hypothesis that: (1) on exposure to chilling temperature, sucrose content in sugarcane leaves is determined by the photosynthetic rate in the leaves, and is not related to SPS activity; (2) SPS activity in sugarcane leaves at chilling temperature is to be determined by sugar concentration in the leaves.     

Effects of Benzyladenine on Chlorophyll, DNA, RNA and Protein Content of Attached Young Bean (Phaseolus vulgaris L.) Leaves

N⁶-Benzyladenine (BA) was applied to intact bean (Phaseolusvulgaris L.) primary leaves at 2 and 6 days after imbibition,when they were in the cell division and post-cell division stages,respectively. BA treatment at day 2 temporarily inhibited an increase in chlorophyllcontent in the following day, but stimulated it in later days.No such inhibition by BA was observed for changes with timein DNA, RNA, and protein content and f. wt. On the other hand,BA treatment at day 6 enhanced RNA and protein content, withoutsignificant influence on DNA and chlorophyll content and f.wt. The mode of cytokinin action on greening in leaves during cell-divisiongrowth seems to be different from that in etiolated cotyledons. Phaseolus vulgaris L., bean, greening, benzyladenine, DNA, RNA, protein     

The role of proteolytic enzymes in protein degradation during senescence of rice leaves

The role of proteolytic enzymes in protein degradation of detached and intact leaves of rice seedling ( Oryza sativa L. cv. Taiching Native 1) during senescence and of mature leaves during reproductive development was investigated. The amount of soluble protein decreased by about 50% in 2, 4, and 15 days for detached, intact and mature leaves, respectively. Three proteolytic enzyme activities were monitored with pH optima of 4.5 for hemoglobin-digesting proteinase, 5.5 for carboxypeptidase and 8.0 for aminopeptidase. No azocoll-digesting proteinase activity could be detected in rice leaves. Dialysis did not alter the activities of any of the three proteolytic enzymes. Acid proteinase activity and aminopeptidase activity were highly unstable during storage of the enzyme extracts at 4°C. Proteolysis was stimulated by inclusion of meroaptoethanal either in the extraction medium or the assay medium.
Acid proteinase, carboxypeptidase and aminopeptidase were all present in detached, intact and mature leaves throughout senescence. There seems to be a direct correlation between protein degradation and increases of acid proteinase and carboxypeptidase activity in seedling leaves (detached and intact) during senescence. In senescing (detached and intact) leaves of seedlings the acid proteinase activity developed first, while that of carboxypeptidase developed later. Acid proteinase and carboxypeptidase may play major roles in protein degradation of leaves from seedlings during senscence. During reproductive development, protein degradation was associated with decreases in the activities of acid proteinase, carboxypeptidase and aminopeptidase in mature leaves suggesting that the enzymes were less important for protein degradation in this system. Hence, the role of protelytic enzymes in protein degradation during senescence of rice leaves appears to depend largely on the leaf system used.     

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