Supercooling Ability, Water Content and Hardiness of Rhododendron Flower Buds during Cold Acclimation and Deacclimation

The relationship between supercooling ability and water contentand killing temperature of flower buds during cold acclimationand deacclimation were studied using R. kiusianum and R. x akebono.The occurrence of multiple floret exotherms and their shiftto a narrow range at lower subzero temperatures, as well asthe marked decrease of florets water content, were observedas the symptoms of cold acclimation occuring in flower budsfrom fall to winter, and vice versa in spring buds during deacclimation.In R. kiusianum, the fully acclimated period was from Novemberto March and two months longer than that of R. x akebono. Thesupercooling ability of the former was about –25°Cand about –20°C in the latter. Although the watermigration within bud tissues during the freezing process wasdetermined in the acclimated and deacclimated buds for R. xakebono, no significant water changes could be observed, evenin the acclimated buds. Thus, it is conceivable that deep supercoolingin florets may result not necessarily from water migration fromflorets and bud axes to scales in response to freezing, butfrom low water content in situ of cold-acclimated or artificiallydehydrated flower buds. (Received July 29, 1981; Accepted October 12, 1981)     

Characteristics of Cold Acclimation and Deacclimation in Tuber-bearing Solanum Species

The effect of temperatures on cold acclimation and deacclimation in foliage tissues was studied in Solanum commersonii (Oka 4583), a tuber-bearing potato. The threshold temperature for cold acclimation was about 12 C. In a temperature range of 2 to 12 C, the increase in hardiness was dependent on the acclimating temperature; the lower the acclimating temperature, the more hardiness achieved. A day/night temperature of 2 C, regardless of photoperiod, appeared to the optimum acclimating temperature for the Solanum species studied. A subfreezing temperature hardened plants less effectively. The maximum level of hardiness could be reached after 15 days of cold acclimation. However, it took only 1 day to deacclimate the hardened plants to a preacclimation level when plants were subjected to a warm regime from cold. The degree of deacclimation was dependent on the temperature of the warm regime.     

Cold Hardiness and Acclimation Intensity in Flower Buds for Fall Bloom and Spring Bloom Clones in Rhododendron kiusianum, a Dwarf Evergreen Azalea

The relationship between the degree of cold hardiness (supercoolingability of florets) and the acclimation intensity in flowerbuds was investigated in the fall bloom and the spring bloom(typical) clones of Rhododendron kiusianum, a hardy dwarf evergreenazalea. Supercooling ability or exotherm temperature distribution(ETD) in florets was determined by differential thermal analysis(DTA) and the intensity of bud acclimation or the rate of deacclimationwas judged by the changes in ETD profiles resulting from thedehardening temperature treatment. Although the two clone typesshowed no significant differences in ETDs and water contentsin florets, they differed in their rates of bud deacclimation.The flower buds of fall bloom clones generally tend to deacclimatemore quickly than the spring bloom ones throughout the seasons.It is concluded that the degree of cold hardiness in flowerbuds of R. kiusianum does not differ between the fall bloomand spring bloom clones but the intensity of bud acclimationdoes; acclimation intensity is higher in the spring bloom clonesand the rate of deacclimation is greater in the fall bloom ones. (Received October 14, 1985; Accepted February 5, 1986)     

Estimation of the Freezing Injury in Flower Buds of Evergreen Azaleas by Water Proton Nuclear Magnetic Resonance Relaxation Times

Temperature dependence of longitudinal relaxation times (T₁)of water protons in flower buds of six azalea species differingin cold hardiness and ecological distribution was investigatedby pulse nuclear magnetic resonance spectroscopy. Thermal hysteresiswas observed for T₁ following a slow freeze-thaw cycle. TheT₁ ratio (the ratio obtained from the difference between theoriginal T₁ value in an unfrozen sample and the final T₁ aftera freeze-thaw treatment, both at 20C, divided by the originalT₁) was closely correlated with the viability of florets innon-acclimated buds of R. kiusianum. If the buds were frozento a lethal temperature and then thawed to 20C, the T₁ ratioincreased. The T₁ ratios of acclimated winter buds for the sixspecies used were correlated with the level of cold hardiness(supercooling ability of florets determined by differentialthermal analysis). The T₁ ratio of deacclimated spring buds,especially those from hardier species, markedly increased uponcooling to a lethal temperature. Species differences observedin acclimated winter buds disappeared upon deacclimation. TheT₁ ratio appears to be related to the viability of florets andthe degree of freezing damage (membrane disruption) in florets. (Received December 28, 1984; Accepted May 24, 1985)     

Changes in Sugar Content during Cold Acclimation and Deacclimation of Cabbage Seedlings

The relationship between freezing tolerance and sugar contentin cabbage seedlings was investigated. Seedlings exposed tonon-freezing low temperature (5 °C) acquired freezing tolerancedown to -6 °C. The degree of freezing tolerance increasedwith duration of exposure to low temperature (up to 10 d). Sucrose,glucose, fructose and myo -inositol were detected as solublesugars in cabbage leaves, and all soluble sugars, except formyo -inositol, and starch increased gradually during cold acclimationsuch that their levels were positively correlated with the degreeof freezing tolerance. The induced freezing tolerance was attributednot to ontogenetic changes but to cold acclimation. However,the induced freezing tolerance was lost after only 1 d of deacclimationat control temperatures, and this change was associated witha large reduction in sugar content. These results reveal that the sugar content of cabbage leavesis positively correlated with freezing tolerance. Brassica oleracea L.; cabbage; cold acclimation; deacclimation; freezing tolerance; sugars     

Relationship of Nuclear Magnetic Resonance Relaxation Time to Water Content and Cold Hardiness in Flower Buds of Evergreen Azalea

The longitudinal relaxation time (T₁) of water protons in floretsof R. ? akebono flower buds was measured with a pulse NMR spectrometerto determine the relationship of T₁ to water content and coldhardiness (supercooling ability). Seasonal changes of T₁ inflorets were closely correlated with water content and supercoolingability of florets. T₁ of florets was short for acclimated budshaving a low water content and long for non-acclimated budshaving a high water content. Flower buds collected in Novemberand stored at 0 and 5?C for 4 weeks had shorter T₁ values thanbuds stored at 10?C even though the floret water content andsupercooling ability were similar. Thus, the short T₁ of coldacclimated buds hardened naturally or by storage at low temperaturesis due to a combination of both reduced water content and temperature. (Received August 27, 1983; Accepted May 26, 1984)     

低温锻炼对冰温贮藏牡丹切花抗冷性的影响

以牡丹品种‘玉面桃花’、‘清香白’和‘凤丹’切花为试验材料,测定室温对照(15~18℃保湿贮藏3d,RTS)、低温锻炼[(4±1)℃保湿贮藏3d,CAS]、冰温贮藏[(4±1)℃保湿贮藏3d,然后转入(-4±1)℃保湿贮藏3d,ITS]3种处理的牡丹切花花枝不同器官(花瓣、花药、子房、萼片、叶柄、叶片、茎杆)的过冷点(SCP)和冰点温度(FP),以及花瓣和叶片束缚水与渗透调节物质含量变化,明确低温锻炼对其切花抗冷能力的影响,为牡丹切花储运及销售过程中冰温贮藏温度控制提供参考依据。结果表明:经4℃低温锻炼3d的牡丹切花与室温对照相比,低温锻炼处理使3个牡丹切花不同组织的SCP和FP显著降低,但花瓣和叶片的束缚水、可溶性蛋白质、可溶性糖和脯氨酸含量均得到提高;冷锻炼后转入冰温贮藏处理进一步提高了牡丹切花花瓣和叶片的束缚水和可溶性蛋白质含量。研究发现,4℃低温锻炼显著提高了牡丹切花的抗冷性,使花枝能够更好地适应30~150d长期贮藏的-3~-4℃冰温环境。     

Group 3 LEA Gene HVA1 Regulation by Cold Acclimation and Deacclimation in Two Barley Cultivars with Varying Freeze Resistance

The level of expression of the group 3 late embryogenesis abundant abscisic acid-regulated gene (HVA1) to cold treatment has been studied in winter barley (Hordeum vulgare) seedling tissue. The cDNA clone (pHVA1) encoding this late embryogenesis abundant protein was used as a hybridization probe to detect the corresponding mRNA. Expression of the HVA1 gene was determined after the tissue had been subjected to a regimen of 2°C exposure (cold acclimation), followed by a return to 25°C growth conditions (deacclimation). Accumulation of HVA1 mRNA occurred upon cold acclimation of the tissue and disappeared as early as 2 hours after exposure to deacclimation conditions. A comparison of the response to cold acclimation and deacclimation was made between seedling tissue of a freeze-resistant and less freeze-resistant cultivar. In both cultivars, the HVA1 gene was expressed and modulated by cold treatment. Within 2 hours of deacclimation HVA1 mRNA was no longer detectable in either cultivar independently of freeze resistance. The level of expression of HVA1 appeared to be greater in the less freeze-resistant cultivar (Winter Malt).     

The Effects of Brown Rust and Phosphate on Cold Acclimation in Winter Barley

The green leaf area of winter barley, cv. Sonja, sampled fromthe field at different times during winter was always greatestin plants grown at high soil phosphate and smallest in plantsgrown at low soil phosphate, and at each fertilizer level wasgreater in healthy plants than in plants infected by rust (Pucciniahordei). In leaves that survived the coldest period of winter,the percentage area that was damaged was increased by rust infectionwhich prevented the ameliorating effects of high soil P. Rustand low P interacted to reduce the increases in leaf area andshoot d. wt that occurred when higher temperatures prevailedin spring. Under controlled conditions in the laboratory, phosphate reducedthe injury suffered when plants not acclimated to low temperatureswere exposed to freezing conditions, but this effect was removedby rust infection. After rust infection, freezing temperatureswere damaging even to acclimated plants, particularly if grownwith low soil P. Evidence of visible symptoms, and quantitativemeasurements of electrolyte efflux from intact leaves, chlorophyllfluorescence in vivo, and ethane and ethylene evolution fromcold-acclimated plants, showed that infection raised the minimumtemperature at which tissues could survive without injury. Infectedleaves were more sensitive to low temperature post-sporulationthan presporulation. Measurements of electrolyte efflux andchlorophyll fluorescence on plants growing under cold conditionsshowed that infection inhibited the processes of acclimationto low temperatures. Winter barley, Puccinia hordei, injury, low temperature, acclimation     

The Effects of Cold Acclimation on Several Enzyme Activities in Cucumber Seedling

The changes in activities of SOD, peroxidase, catalase and ATPase in chilling sensitive cucumber (Cucumis sativus L.) seedlings using biochemical and cytochemical methods were studied. The results indicated that the activities of SOD, peroxidase and catalase enhanced dramatically in cold acclimated cucumber seedlings and the three enzymes remained stable under chilling stress. Consequently, the ability of cleaning up free radical of oxygen and peroxidates increased. The cold-tolerant character of plasmalemma ATPase activity was developed after low-temperature acclimation. All these changes provided the possibility for protecting the stability of membrane structure and metabolism from chilling injury, and for the enhancement of cold tolerance by low temperature acclimation.     

Variations in Cold Resistance among Apple Cultivars during Deacclimation

Coleman, W. K. 1985. Variations in cold resistance among applecultivars during deacclimation.——J. exp. Bot. 36:1159–1171. One-year-old vegetative twig samples from mature, bearing treesof nine apple cultivars were monitored over two years for theirdormancy intensity and relative cold hardiness levels duringthe winter/spring deacclimation period. The apple cultivarsexhibited a consistent response during the dehardening processwhich included a higher initiation temperature for the low temperatureexotherm (LT₂) and the development of an intermediate freezingexotherm (LT₁.). Imperial Red Mac/Antonovka was the hardiestcultivar during the two-year period while Imperial Red Mac/M.111was the most tender. Cortland/Beautiful Arcade and Rogers RedMac/M.111 varied considerably in their relative hardiness responsesfrom year to year. Mid-winter hardiness levels were significantlyand positively correlated with dormancy intensity in the ninecultivars. However, this relationship did not exist when thehardiness indices for late winter or early spring were comparedwith dormancy intensity. An intensive correlation and path analysisof the response of four cultivars (Jersey Mac/M.111, Vista Bella/M.111,Spur Mac/M.111 and Rogers Red Mac/M.111) to previous maximum/minimumair temperatures indicated that past maximum temperature primarilyaffected LT₂ while past minimum temperature affected LT₁. Whenlinear regression equations were fitted to the data, the meanair temperature of 0°C coincided with LT₁ values of —18 °C and LT₂ values of –36°C to –38°Cfor all four cultivars. Correlation analyses between % moisturecontent and LT₁/LT₂ for the four cultivars were often positivebut generally non-significant. Injury in living cells slightlypreceded the initiation temperature of LT₁ and supports theidea that membrane destabilization may be an important and immediateprecursor to intracellular freezing. Key words: Apple, cold hardiness, deacclimation     

Cold Acclimation in Arabidopsis thaliana

The abilities of two races of Arabidopsis thaliana L. (Heyn), Landsberg erecta and Columbia, to cold harden were examined. Landsberg, grown at 22 to 24°C, increased in freezing tolerance from an initial 50% lethal temperature (LT₅₀) of about −3°C to an LT₅₀ of about −6°C after 24 hours at 4°C; LT₅₀ values of −8 to −10°C were achieved after 8 to 9 days at 4°C. Similar increases in freezing tolerance were obtained with Columbia. In vitro translation of poly(A⁺) RNA isolated from control and cold-treated Columbia showed that low temperature induced changes in the population of translatable mRNAs. An mRNA encoding a polypeptide of about 160 kilodaltons (isoelectric point about 4.5) increased markedly after 12 to 24 h at 4°C, as did mRNAs encoding four polypeptides of about 47 kilodaltons (isoelectric points ranging from 5-5.5). Incubation of Columbia callus tissue at 4°C also resulted in increased levels of the mRNAs encoding the 160 kilodalton polypeptide and at least two of the 47 kilodalton polypeptides. In vivo labeling experiments using Columbia plants and callus tissue indicated that the 160 kilodalton polypeptide was synthesized in the cold and suggested that at least two of the 47 kilodalton polypeptides were produced. Other differences in polypeptide composition were also observed in the in vivo labeling experiments, some of which may be the result of posttranslational modifications of the 160 and 47 kilodalton polypeptides.     

植物冷驯化相关信号机制

植物经过非致死温度的处理可以获得更强的抗冷能力叫做冷驯化,主要包括寒驯化和冻驯化 .在冷驯化过程中,质膜首先感受冷信号,调节胞质中IP3的含量,诱导胞质Ca2+浓度的升高,从而激活CBF基因的表达.至今已经克隆了大量的冷调控基因,组成了复杂的信号传导网络,其中ICE1-CBF-COR通路在植物的冷驯化过程中起到重要的作用.ICE1基因编码一个MYB类型的碱性螺旋 环-螺旋（bHLH）转录因子,在上游调节CBF和 其它转录因子的表达,提高抗冷性. HOS1蛋白通过泛素化介导的蛋白降解负调控ICE1,另外,CBF还通过转录的自我调控保持恰当的表达水平.基因的分析研究证明,RNA修饰和核质转运在植物的抗冷过程中也具有重要作用.在不依赖于CBF的途径中,转录因子HOS9和HOS10在调节抗冷有关基因的表达和提高抗冷能力方面具有至关重要的作用.     

Cold Acclimation of Suspension Cultures of Pinus sylvestris in Response to Light and Temperature Treatments

Suspension cultures of Pinus sylvestris L. (Provenance Södra Ydre) were used to determine frost hardiness after manipulating daylengths and temperatures. Frost hardiness was determined with the triphenyltetrazolium chloride (TTC) reduction method. The cultures were able to acclimate and increase frost hardiness levels; both low temperature (2°C) and short day (8 h) treatments were used at the same time, but increased survival temperatures were not achieved when only one type of stimulus was used. Inasmuch as intact seedlings can be partially acclimated by a single type of stimulus, the results indicate that the organization of the cells to tissues plays a role for the hardening process in vivo.     

Phenotypic Plasticity in Photosynthetic Temperature Acclimation among Crop Species with Different Cold Tolerances

While interspecific variation in the temperature response of photosynthesis is well documented, the underlying physiological mechanisms remain unknown. Moreover, mechanisms related to species-dependent differences in photosynthetic temperature acclimation are unclear. We compared photosynthetic temperature acclimation in 11 crop species differing in their cold tolerance, which were grown at 15°C or 30°C. Cold-tolerant species exhibited a large decrease in optimum temperature for the photosynthetic rate at 360 μL L⁻¹ CO₂ concentration [Opt (A₃₆₀)] when growth temperature decreased from 30°C to 15°C, whereas cold-sensitive species were less plastic in Opt (A₃₆₀). Analysis using the C₃ photosynthesis model shows that the limiting step of A₃₆₀ at the optimum temperature differed between cold-tolerant and cold-sensitive species; ribulose 1,5-bisphosphate carboxylation rate was limiting in cold-tolerant species, while ribulose 1,5-bisphosphate regeneration rate was limiting in cold-sensitive species. Alterations in parameters related to photosynthetic temperature acclimation, including the limiting step of A₃₆₀, leaf nitrogen, and Rubisco contents, were more plastic to growth temperature in cold-tolerant species than in cold-sensitive species. These plastic alterations contributed to the noted growth temperature-dependent changes in Opt (A₃₆₀) in cold-tolerant species. Consequently, cold-tolerant species were able to maintain high A₃₆₀ at 15°C or 30°C, whereas cold-sensitive species were not. We conclude that differences in the plasticity of photosynthetic parameters with respect to growth temperature were responsible for the noted interspecific differences in photosynthetic temperature acclimation between cold-tolerant and cold-sensitive species.The temperature dependence of leaf photosynthetic rate shows considerable variation between plant species and with growth temperature (Berry and Björkman, 1980; Cunningham and Read, 2002; Hikosaka et al., 2006). Plants native to low-temperature environments and those grown at low temperatures generally exhibit higher photosynthetic rates at low temperatures and lower optimum temperatures, compared with plants native to high-temperature environments and those grown at high temperatures (Mooney and Billings, 1961; Slatyer, 1977; Berry and Björkman, 1980; Sage, 2002; Salvucci and Crafts-Brandner, 2004b). For example, the optimum temperature for photosynthesis differs between temperate evergreen species and tropical evergreen species (Hill et al., 1988; Read, 1990; Cunningham and Read, 2002). Such differences have been observed even among ecotypes of the same species (Björkman et al., 1975; Pearcy, 1977; Slatyer, 1977).Temperature dependence of the photosynthetic rate has been analyzed using the biochemical model proposed by Farquhar et al. (1980). This model assumes that the photosynthetic rate (A) is limited by either ribulose 1,5-bisphosphate (RuBP) carboxylation (A_c) or RuBP regeneration (A_r). The optimum temperature for photosynthetic rate in C₃ plants is thus potentially determined by (1) the temperature dependence of A_c, (2) the temperature dependence of A_r, or (3) both, at the colimitation point of A_c and A_r (Fig. 1; Farquhar and von Caemmerer, 1982; Hikosaka et al., 2006).Open in a separate windowFigure 1.A scheme illustrating the shift in the optimum temperature for photosynthesis depending on growth temperature. Based on the C₃ photosynthesis model, the A₃₆₀ (white and black circles) is limited by A_c (solid line) or A_r (broken line). The optimum temperature for the photosynthetic rate is potentially determined by temperature dependence of A_c (A), temperature dependence of A_r (B), or the intersection of the temperature dependences of A_c and A_r (C). When the optimum temperature for the photosynthetic rate shifts to a higher temperature, there are also three possibilities determining the optimum temperature: temperature dependence of A_c (D), temperature dependence of A_r (E), or the intersection of the temperature dependences of A_c and A_r (F). Especially in the case that the optimum temperature is determined by the intersection of the temperature dependences of A_c and A_r, the optimum temperature can shift by changes in the balance between A_c and A_r even when the optimum temperatures for these two partial reactions do not change.In many cases, the photosynthetic rate around the optimum temperature is limited by A_c, and thus the temperature dependence of A_c determines the optimum temperature for the photosynthetic rate (Hikosaka et al., 1999, 2006; Yamori et al., 2005, 2006a, 2006b, 2008; Sage and Kubien, 2007; Sage et al., 2008). As the temperature increases above the optimum, A_c is decreased by increases in photorespiration (Berry and Björkman, 1980; Jordan and Ogren, 1984; von Caemmerer, 2000). Furthermore, it has been suggested that the heat-induced deactivation of Rubisco is involved in the decrease in A_c at high temperature (Law and Crafts-Brandner, 1999; Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004a; Yamori et al., 2006b). Numerous previous studies have shown changes in the temperature dependence of A_c with growth temperature (Hikosaka et al., 1999; Bunce, 2000; Yamori et al., 2005). Also, the temperature sensitivity of Rubisco deactivation may differ between plant species (Salvucci and Crafts-Brandner, 2004b) and with growth temperature (Yamori et al., 2006b), which may explain variation in the optimum temperature for photosynthesis (Fig. 1, A and D).A_r is more responsive to temperature than A_c and often limits photosynthesis at low temperatures (Hikosaka et al., 1999, 2006; Sage and Kubien, 2007; Sage et al., 2008). Recently, several researchers indicated that A_r limits the photosynthetic rate at high temperature (Schrader et al., 2004; Wise et al., 2004; Cen and Sage, 2005; Makino and Sage, 2007). They suggested that the deactivation of Rubisco at high temperatures is not the cause of decreased A_c but a result of limitation by A_r. However, it remains unclear whether limitation by A_r is involved in the variation in the optimum temperature for the photosynthetic rate (Fig. 1, B and E).A shift in the optimum temperature for photosynthesis can result from changes in the balance between A_r and A_c, even when the optimum temperatures for these two partial reactions do not change (Fig. 1, C and F; Farquhar and von Caemmerer, 1982). The balance between A_r and A_c has been shown to change depending on growth temperature (Hikosaka et al., 1999; Hikosaka, 2005; Onoda et al., 2005a; Yamori et al., 2005) and often brings about a shift in the colimitation temperature of A_r and A_c. Furthermore, recent studies have shown that plasticity in this balance differs among species or ecotypes (Onoda et al., 2005b; Atkin et al., 2006; Ishikawa et al., 2007). Plasticity in this balance could explain interspecific variation in the plasticity of photosynthetic temperature dependence (Farquhar and von Caemmerer, 1982; Hikosaka et al., 2006), although there has been no evidence in the previous studies that the optimum temperature for photosynthesis occurs at the colimitation point of A_r and A_c.Temperature tolerance differs between species and, with growth temperature, even within species from the same functional group (Long and Woodward, 1989). Bunce (2000) indicated that the temperature dependences of A_r and A_c to growth temperature were different between species from cool and warm climates and that the balance between A_r and A_c was independent of growth temperature for a given plant species. However, it was not clarified what limited the photosynthetic rate or what parameters were important in temperature acclimation of photosynthesis. Recently, we reported that the extent of temperature homeostasis of leaf respiration and photosynthesis, which is assessed as a ratio of rates measured at their respective growth temperatures, differed depending on the extent of the cold tolerance of the species (Yamori et al., 2009b). Therefore, comparisons of several species with different cold tolerances would provide a new insight into interspecific variation of photosynthetic temperature acclimation and their underlying mechanisms. In this study, we selected 11 herbaceous crop species that differ in their cold tolerance (Yamori et al., 2009b) and grew them at two contrasting temperatures, conducting gas-exchange analyses based on the C₃ photosynthesis model (Farquhar et al., 1980). Based on these results, we addressed the following key questions. (1) Does the plasticity in photosynthetic temperature acclimation differ between cold-sensitive and cold-tolerant species? (2) Does the limiting step of photosynthesis at several leaf temperatures differ between plant species and with growth temperature? (3) What determines the optimum temperature for the photosynthetic rate among A_c, A_r, and the intersection of the temperature dependences of A_c and A_r?     

冷驯化对中缅树鼩产热能力的影响

在 ( 5± 1)℃条件下对中缅树 (Tupaiabelangeri)进行低温胁迫处理 ( 0～ 2 8d) ,测定其冷驯化过程中的静止代谢率 (RMR)、非颤抖性产热 (NSTmax)、冷诱导最大产热 (CIRMR)、体重、体温等生理指标 ,探讨低温对中缅树产热能力的影响。结果表明 :①冷驯化期间中缅树体重增加 ,体温降低 ,产热能力显著增强 ;②在冷驯化过程中 ,增加RMR和CIRMR是中缅树抵抗低温胁迫的主要产热模式。     

Biosynthesis of Caffeine in Flower Buds of Camellia sinensis

The biosynthesis of purine alkaloids in flower buds of tea plantswas investigated. More than 25% of total radioactivity of [8-¹⁴C]adeninetaken up by stamens isolated from tea flower buds was foundto have been incorporated into purine alkaloids, namely, theobromineand caffeine, 24 h after administration of the labelled compound.Pulse-chase experiments indicated that [8-¹⁴C]adenine takenup by the stamens was converted to adenine nucleotides and subsequentlyincorporated into theobromine and caffeine. Since 5 µMcoformycin, an inhibitor of AMP deaminase, inhibited the incorporationof radioactivity into the purine alkaloids, synthesis of caffeinefrom adenine nucleotides seems to be initiated by the reactionof AMP deaminase. Although most of the radioactivity from [8-¹⁴C]inosinewas recovered as CO₂ and ureides, considerable amounts of radioactivitywere recovered as purine alkaloids. The incorporation of radioactivityfrom [8-¹⁴C]inosine into the purine alkaloids was not affectedby coformycin. The five enzymes involved in synthesis of 5-phosphoribosyl-1-pyrophosphatefrom glucose were present in the stamens and petals of tea flowerbuds. From present and previous results, the pathway for thebiosynthesis of caffeine from adenine nucleotides in flowerbuds of tea is discussed.Copyright 1993, 1999 Academic Press Camellia sinensis, tea, stamen, flower, biosynthesis, purine alkaloids, caffeine, theobromine, adenine nucleotides, nucleotide biosynthesis