Changes in the Soluble Protein and Free Amino Acid Content of Chill-Sensitive and Chill-Resistant Plants During Chilling and Hardening Treatments

The effects of low temperature (5 °C and 12°C) and droughttreatments on leaf soluble protein content and free amino acidcontent have been investigated in four species, which were rankedaccording to chilling-sensitivity: pea (chill-resistant), mungbean (highly chill-sensitive), and tomato and french bean (intermediatechilling-sensitivity). Drought treatment caused a 30–40% decrease in proteinlevels, and in all but the mung bean, a 100–200% increasein free amino acid concentration. Four days chilling at 5°C,85% r.h. caused leaf water content to decrease by almost 50%in the mung bean, but by only approximately 6–7% in theother three species. During this treatment the leaf solubleprotein content decreased in all four species although the decreasewas greatest and most rapid in the mung bean, commencing with8 h of chilling (coinciding closely with the onset of waterloss), and decreasing by over 80% after 4 d. In the chill-sensitivespecies (but not in the pea) the decrease in protein contentwas accompanied by an increase in free amino acid content. However,on a mgg^–1 dry wt. basis, this increase was insufficientto account for all the protein lost. When plants of each specieswere chilled at 5°C, 100% r.h., water loss was greatly reducedor prevented and there was no significant decrease in leaf solubleprotein. It is concluded that the protein decrease which occurredat 5°C, 85% r.h., was a response to water loss and not thedirect result of low temperature. However, chilling at 100%r.h. did cause an increase in free amino acid content of thechill-sensitive species, suggesting that this was a direct responseto low temperature. Although drought treatment caused a 6–20 fold increasein free proline content in the leaves of the four species examined,chilling (5°C) and chill-hardening (12°C) caused littlechange in free proline content, indicating that the accumulationof this ‘protective’ amino acid is unlikely to contributeto the effectiveness of the chill-hardening treatment. Key words: Low Temperature, Drought, Leaf soluble protein.content, Amino acids     

The Susceptibility of Cucumber and Sweet Pepper to Chilling Under Low Irradiance is Related to Energy Dissipation and Water-Water Cycle

The photoprotection of energy dissipation and water-water cycle were investigated by comparing chilling sensitivity of photosystems 2 (PS2) and 1 (PS1) in two chilling-sensitive plants, cucumber and sweet pepper, upon exposure to 4 °C under low irradiance (100 μmol m⁻² s⁻¹) for 6 h. During chilling stress, the maximum photochemical efficiency of PS2 (F_v/F_m) decreased only slightly in both plants, but the oxidisable P700 decreased markedly, which indicated that PS1 was more sensitive to chilling treatment under low irradiance than PS2. Sweet pepper leaves had lower F_v/F_m, higher non-photochemical quenching (NPQ), and higher oxidisable P700 during chilling stress. Activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in cucumber leaves was higher, but APX activity decreased apparently compared to that at room temperature. The productions of active oxygen species (H₂O₂, O₂ ⁻) increased in both plants, faster in cucumber leaves than in sweet pepper leaves. In sweet pepper leaves, a stronger de-epoxidation of the xanthophyll cycle pigments, a higher NPQ could act as a major protective mechanism to reduce the formation of active oxygen species during stress. Thus sensitivity of both plants to chilling under low irradiance was dominated by the protective mechanisms between PS1 and PS2, especially the energy dissipation and the water-water cycle. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cold-Inhibited Phloem Translocation in Sugar Beet: II. CHARACTERIZATION AND LOCALIZATION OF THE SLOW-COOLING RESPONSE

Experiments were undertaken on a simplified sugar beet systemto characterize the phloem translocation response to slow coolingtreatments that were applied to the source leaf petiole. Inthese experiments the temperature was decreased by 4°C every16 min, such that the tissue temperature was lowered from 25°Cto 1°C over a period of 80 min. Our results indicated thatan initial slow cooling treatment, on a given test plant, causedno change in the rate of translocation. However, all subsequentslow cooling regimes that were applied to the same petiole positionelicited a characteristic step-type inhibition. This inhibitionaveraged about 10% of the original translocation rate in allcases with no recovery being observed. The data suggest thatthe initial cooling treatment induced an alteration in the petioletissue which facilitated the inhibition phenomenon during subsequentslow coolings. This alteration was shown to be localized withinthe upstream region of the chilled petiole segment, followingan initial slow cooling, or throughout the chilled petiole segmentafter an initial quick cooling from 25°C to 1°C. Resultsalso show that the alteration is a long-lived phenomenon thathas no detectable influence on the quick-cooling induced transientinhibition of translocation. Key words: Phloem, Translocation, Cooling response, Petiole     

Synergistic action of rapid chilling and nisin on the inactivation of Escherichia coli

The effect of rapid and slow chilling on survival and nisin sensitivity was investigated in Escherichia coli. Membrane permeabilization induced by cold shock was assessed by uptake of the fluorescent dye 1-N-phenylnapthylamine. Slow chilling (2°C min⁻¹) did not induce transient susceptibility to nisin. Combining rapid chilling (2,000°C min⁻¹) and nisin causes a dose-dependent reduction in the population of cells in both exponential and stationary growth phases. A reduction of 6 log of exponentially growing cells was achieved with rapid chilling in the presence of 100 IU ml⁻¹ nisin. Cells were more sensitive if nisin was present during stress. Nevertheless, addition of nisin to cell suspension after the rapid chilling produced up to 5 log of cell inactivation for exponentially growing cells and 1 log for stationary growing cells. This suggests that the rapid chilling strongly damaged the cell membrane by disrupting the outer membrane barrier, allowing the sensitization of E. coli to nisin post-rapid chilling. Measurements of membrane permeabilization showed a good correlation between the membrane alteration and nisin sensitivity. Application involving the simultaneous treatment with nisin and rapid cold shock could thus be of value in controlling Gram negatives, enhancing microbiological safety and stability.     

The chilling sensitivity of root ammonium influx in a cultivated and wild tomato

A chilling episode of a few hours damaged root ammonium absorption in a cultivated tomato ( Lycopersicon esculentum cv. T-5), but not in a wild congener from high altitudes ( Lycopersicon hirsutum LA1778). In the cultivar, ammonium influx was strongly temperature dependent and showed the residual effects of chilling, whereas ammonium efflux was nearly temperature invariant and showed no persistent effects. A 2 h exposure to 5 °C significantly depressed subsequent ammonium absorption at 20 °C, and about 12 h at 20 °C was required for recovery. For both the cultivated and wild species, rerooted cuttings were slightly less sensitive to chilling than seedlings. The relative inhibition (mean ± SE) of ammonium absorption before and after chilling was 58·4 ± 2·5% for the cultivated species and 29·0 ± 9·1% for the wild species. The F₁ hybrid between the species showed a relative inhibition of 52·4 ± 3·6%, suggesting that chilling sensitivity may be dominant. In a backcross of the hybrid to L. esculentum , the phenotypic distribution of the relative inhibition of ammonium absorption indicated that this trait is segregating.     

A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism

A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18°C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury—leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of ¹⁴CO₂ into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury. These results suggest one of two possible explanations: either the mutation directly affects sterol metabolism, which in turn leads to chilling sensitivity, or the mutation affects another unidentified function and the accumulation of radioactivity in steryl-esters is a secondary consequence of chilling injury.     

Different root low temperature response of two maize genotypes differing in chilling sensitivity

Here we report on the root hydraulic properties of intact and excised root systems of two maize genotypes differing in chilling sensitivity (Z7, tolerant and Penjalinan, sensitive) subjected for 3 d to 5 °C. When root hydraulic conductance (L) was measured under a hydrostatic force using an excised root system in a pressure chamber, an initial decrease of L was observed in both genotypes. However, the value of L increased in the chilling tolerant genotype after 30 h at 5 °C; in the chilling sensitive Penjalinan genotype there was no such increase. Osmotic root hydraulic conductance was measured in excised root systems exuding under atmospheric pressure. We observed a progressive decline during the chilling treatment of the osmotic root hydraulic conductance in the chilling sensitive Penjalinan plants; however, after 54 h at 5 °C, the chilling tolerant Z7 plants had a significantly higher osmotic hydraulic conductance. Moreover, in the chilling tolerant plants we found an increase in the inhibition caused by HgCl₂ of the osmotic hydraulic conductance during the chilling treatment, indicating a possible increase in the contribution of aquaporins to root hydraulic conductance in the chilling tolerant Z7 plants during chilling treatment.     

Long-Term Chilling of Young Tomato Plants under Low Light. III. Leaf Development as Reflected by Photosynthesis Parameters

Young tomato plants were exposed to two weeks of chilling undernon-photoinhibiting or mild photoinhibiting conditions. Thedevelopment of the leaves was studied under chilling and controlconditions by measuring several physiological parameters. Agradual decrease of the efficiency of the photosynthetic apparatuswith maturation and ageing occurred in unchilled plants. Thiswas reflected by gradual changes in CO₂-saturated photosynthesisand protein and rubisco contents. Except for senescing leaves,a correlation close to 1 : 1 was observed between maximum rubiscoactivity and CO₂-saturated photosynthesis. Chlorophyll (Chl)contents and photochemical chlorophyll fluorescence quenchingshowed strong decreases only in the last phase of senescencein the oldest leaves. In plants chilled under non-photoinhibitingconditions (10C, 100–150 µE m^–2 s^–1or 6C, 30–50 µE m^-2 s^–1), a similar patternof ageing was observed, and no indications were found for aninduction of protein or rubisco degradation by chilling. Sincethese plants stopped growing in the cold, they revealed lowertotal photosynthetic capacities than unchilled plants of thesame size. When the chilling conditions were mildly photoinhibitory(6C, 100–150 µE m^–2 s^–1), a much strongerdepression of rubisco activity and photosynthetic capacity wasfound in all leaves, which was partly reversible in the youngones. This decrease in CO₂fixation capacity, in turn, led toa higher susceptibility of the chilled plants to photoinhibitionat 20C. It is concluded that the decrease of both photosyntheticcapacity and growth after long-term chilling in tomato is aconsequence of the preceeding ageing and senescing of the leavesduring chilling, in contrast to chilling-tolerant species withthe ability for acclimation to low temperatures. (Received April 26, 1993; Accepted September 7, 1993)     

Proline and Polyamine Involvement in Chilling Tolerance of Maize Suspension Cultures

We have assessed the effect of various medium supplements inpromoting the ability of maize (Zea mays L.) inbred FR27rhmsuspension cultures to grow following a period of 4 °C chillingstress. Following a 4 week exposure to 4 °C in culture mediumwithout proline, no cell growth occurred upon subsequent incubationat 28°C for 2 weeks. This inhibition was reversed when 3to 48 mol m^–3 proline or 0.1 mol m^–3 putrescineor 0.01 mol m^–3 spermidine were present in the mediumduring the chilling stress. On the other hand, suspensions weremade more sensitive to 4°C by blocking polyamine biosynthesiswith 1.0 mol m^–3 methylglyoxal bis (guanylhydrazone) (MGBG)or a combination of 1.0 mol m^–3 difluoromethylornithine(DFMO) and 1.0 mol m^–3 difluoromethylarginine (DFMA).The addition of 10 mol m^–3 putrescine to the suspensioncontaining DFMO and DFMA prevented the increased chilling sensitivity.Electrolyte leakage studies conducted to assess membrane integrityafter 4 weeks at 4°C and a 2 week regrowth period showedthat cells treated with no polyamines (control), 0.01 mol m^–3spermidine, 1.0 mol m^–3 putrescine, or 1.0 mol m^–3MGBG lost 43, 32, 14, and 100% of the total electrolyte pool,respectively. These results suggest that proline and polyaminesare beneficial for inducing chilling tolerance in FR27rhm suspension. Key words: Proline, polyamine, chilling stress     

Association of polyamines in governing the chilling sensitivity of maize genotypes

Chilling stress is an important constraint of global production of maize. This study was undertaken to compare the chilling responses of different maize seedling tissues and to analyze changes in polyamines as a result of chilling stress. Reponses to chilling were characterized in two maize (Zea mays L.) inbred lines, ‘HuangC’ and ‘Mo17’, that putatively differ in chilling sensitivity. Seedlings were exposed to low temperature (5°C) and chilling injury was estimated by electrical conductivity (EC), malonaldehyde (MDA) concentration, and by changes in putrescine (Put), spermidine (Spd) and spermine (Spm) concentrations in root, mesocotyl, and coleoptile tissues. Membrane permeability (as measured by EC), MDA concentrations and Put concentrations in the three tissue of maize seedlings increased after chilling stress, except for the Put concentration in roots. Spd and Spm concentrations in the three tissues of seedlings decreased after chilling stress. The EC for cold stressed tissues were lower in HuangC than Mo17. Also, the EC of coleoptile tissues were lower than for mesocotyl in both inbred lines. We suggest that mesocotyl tissue can be used to evaluate cold tolerance in maize. Stepwise regression analyses showed that chilling injury in roots was generally correlated with Spd concentration while in the mesocotyl injury was mainly correlated with Put and Spd concentrations. Spermidine and Spm concentrations in the coleoptile were correlated with chilling injury. Characteristics changes of polyamines in chill-tolerant maize seedling combined with regression analysis are a reliable method for evaluating chill tolerance in maize lines.     

Factors controlling Flowering in the Chrysanthemum: IV. THE SITE OF VERNALIZATION AND TRANSLOCATION OF THE STIMULUS

By confining low-temperature treatment to the growing tip ofthe Chrysanthemum plant, the apex has been shown to be the seatof perception of vernalization, a result which is in full accordwith earlier experiments on other plants requiring vernalization.Experiments on the translocation of the stimulus through maturetissues involving stock/scion and ‘approach’ graftswith or without defoliation gave negative results. By decapitatingin turn the main axis and the resulting laterals produced, ithas been shown that the stimulus is passed on from existingapices to laterals of up to the seventh order at least, lateralapices which were formed well after the time of chilling. Theseresults are discussed in relation to prior work on the existenceof a specific vernalization substance as well as a floweringhormone and their translocation.     

The different effects of chilling stress under moderate light intensity on photosystem II compared with photosystem I and subsequent recovery in tropical tree species

Tropical plants are sensitive to chilling temperatures above zero but it is still unclear whether photosystem I (PSI) or photosystem II (PSII) of tropical plants is mainly affected by chilling temperatures. In this study, the effect of 4°C associated with various light densities on PSII and PSI was studied in the potted seedlings of four tropical evergreen tree species grown in an open field, Khaya ivorensis, Pometia tomentosa, Dalbergia odorifera, and Erythrophleum guineense. After 8 h chilling exposure at the different photosynthetic flux densities of 20, 50, 100, 150 μmol m⁻² s⁻¹, the maximum quantum yield of PSII (F _v /F _m) in all of the four species decreased little, while the quantity of efficient PSI complex (P _m) remained stable in all species except E. guineense. However, after chilling exposure under 250 μmol m⁻² s⁻¹ for 24 h, F _v /F _m was severely photoinhibited in all species whereas P _m was relative stable in all plants except E. guineense. At the chilling temperature of 4°C, electron transport from PSII to PSI was blocked because of excessive reduction of primary electron acceptor of PSII. F _v /F _m in these species except E. guineense recovered to ~90% after 8 h recovery in low light, suggesting the dependence of the recovery of PSII on moderate PSI and/or PSII activity. These results suggest that PSII is more sensitive to chilling temperature under the moderate light than PSI in tropical trees, and the photoinhibition of PSII and closure of PSII reaction centers can serve to protect PSI.     

Interaction between light and chilling temperature on the inhibition of photosynthesis in chilling-sensitive plants*

Abstract. Fully expanded leaves of 25°C grown Phaseolus vulgaris and six other species were exposed for 3 h to chilling temperatures at photon flux densities equivalent to full sunlight. In four of the species this treatment resulted in substantial inhibition of the subsequent quantum yield of CO₂ uptake, indicating reduction of the photochemical efficiency of photosynthesis. The extent of inhibition was dependent on the photon flux density during chilling and no inhibition occurred when chilling occurred at a low photon flux density. No inhibition occurred at temperatures above 11.5°C, even in the presence of the equivalent of full sunlight. This interaction between chilling and light to cause inhibition of photosynthesis was promoted by the presence of oxygen at normal air partial pressures and was unaffected by the CO₂ partial pressure present when chilling occurred in air. When chilling occurred at low O₂ partial pressures, CO₂ was effective in reducing the degree of inhibition. Apparently, when leaves of chilling-sensitive plants are exposed to chilling temperatures in air of normal composition then light is instrumental in inducing rapid damage to the photochemical efficiency of photosynthesis.     

Effects of chilling on the biochemical and functional properties of thylakoid membranes

The mechanism of chilling resistance was investigated in 4-week-old plants of the chilling-sensitive cultivated tomato, Lycopersicon esculentum Mill. cv H722, and rooted cuttings of its chilling-resistant wild relative, L. hirsutum Humb. and Bonpl., which were chilled for 3 days at 2°C with a 14-hour photoperiod and light intensity of 250 micromoles per square meter per second. This chilling stress reduced the chlorophyll fluorescence ratio, stomatal conductance, and dry matter accumulation more in the sensitive L. esculentum than in the resistant L. hirsutum. Photosynthetic CO₂ uptake at the end of the chilling treatment was reduced more in the resistant L. hirsutum than in L. esculentum, but recovered at a faster rate when the plants were returned to 25°C. The reduction of the spin trap, Tiron, by isolated thylakoids at 750 micromoles per square meter per second light intensity was taken as a relative indication of the tendency for the thylakoids to produce activated oxygen. Thylakoids isolated from the resistant L. hirsutum with or without chilling treatment were essentially similar, whereas those from chilled leaves of L. esculentum reduced more Tiron than the nonchilled controls. Whole chain photosynthetic electron transport was measured on thylakoids isolated from chilled and control leaves of the two species at a range of assay temperatures from 5 to 25°C. In both species, electron transport of the thylakoids from chilled leaves was lower than the controls when measured at 25°C, and electron transport declined as the assay temperature was reduced. However, the temperature sensitivity of thylakoids from chilled L. esculentum was altered such that at all temperatures below 20°C, the rate of electron transport exceeded the control values. In contrast, the thylakoids from chilled L. hirsutum maintained their temperature sensitivity, and the electron transport rates were proportionately reduced at all temperatures. This sublethal chilling stress caused no significant changes in thylakoid galactolipid, phospholipid, or protein levels in either species. Nonchilled thylakoid membranes from L. hirsutum had fourfold higher levels of the fatty acid 16:1, than those from L. esculentum. Chilling caused retailoring of the acyl chains in L. hirsutum but not in L. esculentum. The chilling resistance of L. hirsutum may be related to an ability to reduce the potential for free radical production by close regulation of electron transport within the chloroplast.     

Transcript profiling in Vitis riparia during chilling requirement fulfillment reveals coordination of gene expression patterns with optimized bud break

Endodormant grapevine buds require a period of chilling before they break and begin to grow. Custom Vitis bud cDNA microarrays (9,216 features) were used to examine gene expression patterns in overwintering Vitis riparia buds during 2,000 h of 4°C chilling. Three-node cuttings collected concurrently with buds were monitored to determine dormancy status. Chilling requirement was fulfilled after 1,500 h of chilling; however, 2,000 h of chilling significantly increased the rate of bud break. Microarray analysis identified 1,469 significantly differentially expressed (p value < 0.05) array features when 1,000, 1,500, and 2,000 h of chilling were compared to 500 h of chilling. Functional classification revealed that the majority of genes were involved in metabolism, cell defense/stress response, and genetic information processing. The number of significantly differentially expressed genes increased with chilling hour accumulation. The expression of a group of 130 genes constantly decreased during the chilling period. Up-regulated genes were not detected until the later stages of chilling accumulation. Hierarchical clustering of non-redundant expressed sequence tags revealed inhibition of genes involved in carbohydrate and energy metabolism and activation of genes involved in signaling and cell growth. Clusters with expression patterns associated with increased chilling and bud break were identified, indicating several candidate genes that may serve as indicators of bud chilling requirement fulfillment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Low-temperature induced changes in the ultrastructure of maize mesophyll chloroplasts strongly depend on the chilling pattern/intensity and considerably differ among inbred and hybrid genotypes

The ultrastructure and dimensions of chloroplasts in leaf mesophyll cells were quantitatively examined in three parental inbred lines of maize (Zea mays L.) and their four hybrids subjected to two types of four-week low-temperature (LT) treatment: the abrupt onset of chilling temperatures (“severe chilling”, SC) and the gradual, more moderate one (“moderate chilling”, MC). The relationship between the response of individual genotypes to one or the other type of chilling was analyzed as well as the possibility to predict the behaviour of chloroplasts in hybrids from that of their parents. Although selected parameters of chloroplast ultrastructure (e.g. volume densities of granal and intergranal thylakoids, plastoglobuli, and peripheral reticulum) and dimensions changed due to the exposure of maize plants to LT, no general pattern of such changes was found for this species due to the observed intraspecific variability. The response of some genotype to SC could not be predicted from its behaviour under MC (and vice versa) and no clear rules could be applied for the inheritance of chloroplast response to chilling in the general sense. Thus, great caution should be always taken when interpreting the results of studies aimed at the dissection of chloroplast ultrastructure as affected by LT, particularly in case such studies are made with one genotype or under one type of chilling only.     

The mitigating role of environmental factors in seedling injury and chill-dependent depression of catalase activity in maize leaves

In pot experiments performed on maize seedlings chilled at 5 °C, leaf injury was diminished by the application of elevated temperature (1 or 5 h at 15 or 20°C, “warm breaks” treatment) in a dose-dependent manner. The lower the injury count, the higher the catalase (CAT) activity. In a separate experiment, the application of 100 % relative humidity also protected the plants from chilling injury and water loss, increased their gas exchange and variable to maximum chlorophyll fluorescence ratio (F_v/F_m), but did not influence CAT activity. Another protective environmental factor, elevated atmospheric CO₂ concentration [700 μmol(CO₂) mol⁻¹(air)] diminished CAT activity inhibition, but only in plants of chilling-resistant cultivar. The positive impact of specific environmental factors accompanying chilling is not obviously related to the suppression of the inhibition of CAT activity, although the enzyme is considered as chilling-sensitive.     

Differences between the effects of partial and whole plant chilling on carbon translocation of a C4 grass

Abstract Experiments were conducted with Echinochloa crus-galli to partition the effects of chilling the leaf vs. chilling the whole plant on subsequent ¹¹C translocation. The results clearly demonstrated that whole plant chilling was very detrimental whereas chilling only the leaf had no effect on subsequent translocation nor on ¹¹C uptake. The inhibition of translocation was due to a reduced rate and percentage of export while ¹¹C fixation rate was not significantly altered. When the leaf of a chilled plant was maintained at 22 °C, there was no impairment of the transport system nor of photosynthesis. The decrease in export with whole plant chilling may have been due to carbon movement into storage carbohydrates, resulting in a low sucrose gradient.     

Dormancy in Dioscorea: Range, Duration and Timing of High-Temperature Treatment in Germination Inhibition of D. tokoro Seeds

The effects of temperature on induction and release of high-temperatureinhibition in seed germination of Dioscorea tokoro Makino, amonocotyledonous summer perennial of the temperate zone of EastAsia, were investigated. Germination was increasingly inhibitedwith elevation of temperature over 23°C and lengtheningof its duration. The low temperature limit for germination inhibitiondecreased with lengthening of the duration of high temperature.The most sensitive phase for high temperature was 1–2days after the start of imbibition at 20°C. The germination inhibition by high temperature was reversedby chilling at 5°C, which is the optimum temperature forbreaking the natural dormancy (primary dormancy) of this seed.This showed that the high-temperature inhibition of germinationdoes not cause mortal damage but only secondary dormancy (induceddormancy). Seeds from a cold climate (Miyagi Pref.) responded rather quicklyto both high temperature and chilling compared to seeds froma warm climate (Kagoshima Pref.). The responsiveness to hightemperature and chilling of D. tokoro seed may affect the germinationtime under natural conditions. (Received October 22, 1982; Accepted January 14, 1983)