Differential response to paraquat induced oxidative stress in two rice cultivars on antioxidants and chlorophyll a fluorescence

The responses of antioxidative system and photosystem II photochemistry of rice (Oryza sativa L.) to paraquat induced oxidative stress were investigated in a chilling-tolerant cultivar Xiangnuo no. 1, and a chilling-susceptible cultivar, IR-50. Electrolyte leakage and malondialdehyde (MDA) content of Xiangnuo no. 1 were little affected by paraquat, but they increased in IR-50. After paraquat treatment, superoxide dismutase (SOD) activity remained high in Xiangnuo no. 1, while it declined in IR-50. Activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) declined with oxidative stress in both cultivars, but Xiangnuo no. 1 had higher GR activity than IR-50. Under paraquat induced oxidative stress, ascorbic acid (AsA) and reduced glutathione (GSH) concentrations remained high in Xiangnuo no. 1, but decreased in IR-50. The results indicated that higher activities of SOD and GR and higher contents of AsA and GSH in Xiangnuo no. 1 under paraquat induced oxidative stress were associated with its tolerance to paraquat, while paraquat induced damage to IR-50 was related to decreased activities of SOD, APX and GR and contents of AsA and GSH. F _v/F _m, Φ _PSII, and qP remained high in Xiangnuo no. 1, while they decreased greatly in IR-50 under paraquat induced oxidative stress.     

夜间低温对生长在两种光强下两个芒果品种的气体交换和叶绿素荧光的影响

研究了夜间低温对两个芒果(Mangifera indica)品种翡翠芒(Khieo Sawoei)和四季芒(Choke Anand)光合生理的影响.两个芒果品种的幼茼盆栽于全光和50%相对光强下一年.在第二年的冬季,连续7天晚上将芒果幼苗移到4℃的冷库中,白大保持原条件.于低温处理前、处理期间和结束低温处理后10天中测定芒果幼苗的光合生理特征.结果表明,夜间低温导致两个芒果品种的净光合速率、气孔导度和光系统Ⅱ的最大光化学效率(Fv/Fm)降低、非光化学猝灭(NPQ)上升.夜间低温对生长在全光下的芒果幼苗光合作用的抑制比50%光下的更重.翡翠芒的Fv/Fm比四季芒下降的更多,但后者的NPQ上升更多.夜间低温还导致两种光下芒果幼苗叶片的叶绿素含量下降,类胡萝卜素/叶绿素比值、丙二醛含量、膜的透性和可溶性化合物(可溶性总糖和脯氨酸)上升.解除低温胁迫后,四季芒Fv/Fm的恢复比翡翠芒的快.解除低温胁迫7天后二者的F发、Fv/Fm能完全恢复.上述结果表明,翡翠芒对低温更敏感,遮荫可以明显缓解两个芒果品种低温引起的光抑制.     

Impact of salicylic acid on the antioxidant enzyme system and hydrogen peroxide production in Cucumis sativus under chilling stress

Salicylic acid (SA) is a naturally produced compound and has been implicated to play important roles in defense of plants against diverse biotic and abiotic stresses. To understand how SA functions in the tolerance of cucumber (Cucumis sativus) to chilling stress, endogenous SA levels in two different cultivars with opposite chilling responsiveness were quantified. Membrane integrity, including malondialdehyde (MDA) content and leakage of electrolyte, was also examined in SA-pretreated cucumber plants under chilling conditions. In addition, activities of the two antioxidant enzymes peroxidase (POD) and catalase (CAT) were quantified, and hydrogen peroxide (H2O2) production was investigated histochemically in SA-treated leaves under chilling temperature. Chilling stress resulted in greater induction of SA levels in the chilling-tolerant cultivar Changchun mici in both leaves and seeds compared to the chilling-sensitive one Beijing jietou, while the former one contained higher levels of SA than the latter one in the seeds under normal conditions. Pretreatment with SA diminished the increased electrolyte leakage and MDA content caused by chilling in the leaves of both cultivars, while much less MDA and electrolyte leakage were produced in Changchun mici compared to Beijing jietou. Moreover, exogenous application of SA increased significantly the POD and CAT activities and soluble protein content. Most importantly, exogenous SA treatment could eliminate the accumulation of H202 in leaves and cotyledons of both cultivars caused by chilling stress. The data clearly demonstrated that the chilling-tolerant cultivar displays a higher SA level than the chilling-sensitive one, and that exogenous SA can enhance the chilling tolerance ability, which might be achieved through modulating the antioxidant system in cucumber.     

Characteristics of chlorophyll fluorescence and antioxidative system in super-hybrid rice and its parental cultivars under chilling stress

Characteristics of chlorophyll fluorescence and antioxidative system were investigated in rice (Oryza sativa L.) super-hybrid Liangyoupeijiu (LYPJ), maternal cultivar Peiai64s, and paternal cultivar indica rice 9311 under chilling stress. During 6-d chilling treatment, chlorophyll content of all three genotypes was gradually declined. However, the decrease in photosystem 2 (PS 2) maximum photochemical efficiency (F_v/F_m) and quantum yield of PS 2 (Φ_PS2) was less expressive in LYPJ than in parental cultivars The electrolyte leakage and malondialdehyde content in all cultivars increased after chilling treatment, but LYPJ exhibited the least increasing tendency. Activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) were higher in LYPJ than in parental cultivars. The results demonstrated that tolerance to chilling stress in LYPJ might be adopted mostly from its maternal cultivar.     

Comparative lipid peroxidation,antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance

The changes in the activity of antioxidant enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), peroxidase (POX: EC 1.11.1.7), ascorbate peroxidase (APOX: EC 1.11.1.11) and glutathione reductase (GR: EC 1.6.4.2), free proline content, and the rate of lipid peroxidation level in terms of malondialdehyde (MDA) in roots of two rice cultivars (cvs.) differing in salt tolerance were investigated. Plants were subjected to three salt treatments, 0, 60, and 120 mol m⁻³ NaCl for 7 days. The results showed that activated oxygen species may play a role in cellular toxicity of NaCl and indicated differences in activation of antioxidant defense systems between the two cvs. The roots of both cultivars showed a decrease in GR activity with increase in salinity. CAT and APOX activities increased with increasing salt stress in roots of salt-tolerant cultivar Pokkali but decreased and showed no change, respectively, in roots of IR-28 cultivar. POX activity decreased with increasing NaCl concentrations in salt-tolerant Pokkali but increased in IR-28. SOD activity showed no change in roots of both cultivars under increasing salinity. MDA level in the roots increased under salt stress in sensitive IR-28 but showed no change in Pokkali. IR-28 produced higher amount of proline under salt stress than in Pokkali. Increasing NaCl concentration caused a reduction in root fresh weight of Pokkali and root dry weight of IR-28. The results indicate that improved tolerance to salt stress in root tissues of rice plants may be accomplished by increased capacity of antioxidative system.     

Increased Rubisco content in maize mitigates chilling stress and speeds recovery

Many C₄ plants, including maize, perform poorly under chilling conditions. This phenomenon has been linked in part to decreased Rubisco abundance at lower temperatures. An exception to this is chilling‐tolerant Miscanthus, which is able to maintain Rubisco protein content under such conditions. The goal of this study was to investigate whether increasing Rubisco content in maize could improve performance during or following chilling stress. Here, we demonstrate that transgenic lines overexpressing Rubisco large and small subunits and the Rubisco assembly factor RAF1 (RAF1‐LSSS), which have increased Rubisco content and growth under control conditions, maintain increased Rubisco content and growth during chilling stress. RAF1‐LSSS plants exhibited 12% higher CO₂ assimilation relative to nontransgenic controls under control growth conditions, and a 17% differential after 2 weeks of chilling stress, although assimilation rates of all genotypes were ~50% lower in chilling conditions. Chlorophyll fluorescence measurements showed RAF1‐LSSS and WT plants had similar rates of photochemical quenching during chilling, suggesting Rubisco may not be the primary limiting factor that leads to poor performance in maize under chilling conditions. In contrast, RAF1‐LSSS had improved photochemical quenching before and after chilling stress, suggesting that increased Rubisco may help plants recover faster from chilling conditions. Relatively increased leaf area, dry weight and plant height observed before chilling in RAF1‐LSSS were also maintained during chilling. Together, these results demonstrate that an increase in Rubisco content allows maize plants to better cope with chilling stress and also improves their subsequent recovery, yet additional modifications are required to engineer chilling tolerance in maize.     

低温弱光胁迫对野生大豆和大豆栽培种光系统功能的影响

以野生大豆和栽培大豆为材料,通过同时测定大豆叶片的叶绿素荧光快速诱导动力学曲线和对820nm光的吸收曲线,以及测定超氧化物歧化酶（SOD）和抗坏血酸过氧化物酶（APX）的活性,分析了低温弱光胁迫及常温弱光恢复下这2种大豆光系统Ⅱ（PSⅡ）和光系统Ⅰ（PSI）功能的变化。结果表明,低温弱光胁迫对这2种大豆的PSI和PSⅡ的功能都造成伤害;在低温弱光胁迫下,维持较高的SOD和APX活性和维持PSI和PSⅡ的协调性是野生大豆比栽培大豆耐低温的一个重要原因。     

Effects of 24-epibrassinolide on seed germination, seedling growth, lipid peroxidation, proline content and antioxidative system of rice (Oryza sativa L.) under salinity stress

The effects of 24-epibrassinolide (24-epiBL) on seedling growth, antioxidative system, lipid peroxidation, proline and soluble protein content were investigated in seedlings of the salt-sensitive rice cultivar IR-28. Seedling growth of rice plants was improved by 24-epiBL treatment under salt stress conditions. When seedlings treated with 24-epiBL were subjected to 120 mM NaCl stress, the activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and glutathione reductase (EC 1.6.4.2) did not show significant difference, whereas the activity of ascorbate peroxidase (EC 1.11.1.11) significantly increased. Increased activity of peroxidase (EC 1.11.1.7) under NaCl stress showed remarkable decrease in the 24-epiBL+NaCl-applied group. Lipid peroxidation level significantly increased under salt stress but decreased with 24-epiBL application revealing that less oxidative damage occurred in this group (24-epiBL+NaCl). In addition, increased proline content in the NaCl-applied group was decreased by 24-epiBL application in the 24-epiBL+NaCl-applied group. Soluble protein content was increased by 24-epiBL application even under NaCl stress, being also higher than control conditions (no 24-epiBL or NaCl treatment). 24-epiBL treatment considerably alleviated oxidative damage that occurred under NaCl-stressed conditions and improved seedling growth in part under salt stress in sensitive IR-28 seedlings.     

Heat and chilling induced disruption of redox homeostasis and its regulation by hydrogen peroxide in germinating rice seeds (Oryza sativa L., Cultivar Ratna)

Extremes of temperature (both heat and chilling) during early inbibitional phase of germination caused disruption of redox-homeostasis by increasing accumulation of reactive oxygen species (superoxide and hydrogen peroxide) and significant reduction of antioxidative defense (assessed in terms of total thiol content and activities of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) in germinating tissues of rice (Oryza sativa L., cultivar Ratna). Imbibitional heat and chilling stress also induced oxidative damage to newly assembled membrane system by aggravating membrane lipid peroxidation and protein oxidation [measured in terms of thiobarbituric acid reactive substances (TBARS), free carbonyl content (C = O groups) and membrane protein thiol level (MPTL)]. Treatment with standardized low titer hydrogen peroxide during early imbibitional phase of germination caused significant reversal in oxidative damages to the newly assembled membrane system imposed by heat and chilling stress [evident from the data of TBARS, C = O, MPTL, ROS accumulation, membrane permeability status, membrane injury index and oxidative stress index] in seedlings of experimental rice cultivar. Imbibitional H₂O₂ pretreatment also caused up-regulation of antioxidative defense (activities of superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and total thiol content) in the heat and chilling stress-raised rice seedlings. When the parameters of early growth performances were assessed (in terms of relative growth index, biomass accumulation, relative germination performance, mean daily germination, T₅₀ value), it clearly exhibited significant improvement of early growth performances of the experimental rice cultivar. The result proposes that an ‘inductive pulse’ of H₂O₂ is required to switch on some stress acclimatory metabolism through which plant restores redox homeostasis and prevents or repairs oxidative damages to newly assembled membrane system caused by unfavorable environmental cues during early germination to the rice cultivar Ratna. The importance of mitigating oxidative damages to membrane lipid and protein necessary for post-germinative growth under extremes of temperature is also suggested.     

Role of abscisic acid in chilling tolerance of rice (Oryza sativa L.) seedlings. I. Endogenous abscisic acid levels

Changes of ABA levels in chilled rice (Oryza sativa L.) seedlings of two varieties were determined. On exposure to chilling, ABA concentration rapidly increased in the chilling-tolerant cultivar (cv. Tainung 67, TNG.67) but not in the chilling-sensitive cultivar (cv. Taichung Native 1, TN.1). Both detached shoots and roots of TNG.67 seedlings showed a significant ABA increase after exposure to chilling. TN.1 seedlings could not accumulate ABA under low temperature but well-watered status. Exogenous application of the ABA biosynthetic inhibitor, fluridone, reduced ABA accumulation, as well as survival ratio of chilled TNG.67 seedlings. Electrolyte leakage and leaf conductance were also increased by the inhibitor and the effects could be reversed by exogenously applied ABA. ABA concentrations in xylem sap of TNG.67 seedlings increased within 4 h after chilling, and this was temporally coincident with the reduction of leaf conductance. The roles of endogenous ABA in the tolerance of rice seedlings to chilling on a whole plant basis are discussed and suggested.     

Increased Chilling Tolerance Following Transfer of a betA Gene Enhancing Glycinebetaine Synthesis in Cotton (Gossypium hirsutum L.)

A betA gene encoding choline dehydrogenase from Escherichia coli was transformed into cotton (Gossypium hirsutum L.) via Agrobacterium-mediated transformation. Transgenic cotton plants exhibited improved tolerance to chilling due to accumulation of glycinebetaine (GB). The results of our experiment showed that GB contents of leaves of transgenic lines 1, 3, 4, and 5, both before and after chilling stress, were significantly higher than those of wild-type (WT) plants. At 15°C, transgenic lines 1, 3, 4, and 5 exhibited higher germination capacity as determined by the germination speed and final germination percentage and, displayed less inhibition in seedling shoot growth rate than WT plants. Under chilling stress, transgenic lines 4 and 5 maintained higher relative water content, upper carbon dioxide (CO₂) fixation capacity and PSII electron transfer rate, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation when compared with WT plants. Chilling resistance of the transgenic lines was demonstrated to be positively correlated with GB content under chilling stress. The high levels of GB in transgenic cotton plants might not only protect the integrity of cell membrane from chilling damage, but also be involved in OA which alleviated chilling induced water stress. Moreover, under chilling-stressed conditions, transgenic cotton plants enhanced stomatal conductance, PSII electron transport rate, and further leaf photosynthesis through accumulating high levels of GB.     

Exogenous nitric oxide effect on fructan accumulation and FBEs expression in chilling-sensitive and chilling-resistant wheat

This study was to investigate the effect of exogenous nitric oxide (NO) on fructan accumulation and fructan biosynthesic enzymes (FBEs) expression in seedlings leaves of two wheat (Triticum aestivum L.) cultivars, winter wheat (Zhoumai18, ZM) and spring wheat (Yanzhan4110, YZ), under 4 °C. The seedlings of two wheat cultivars were subjected to different concentrations of sodium nitroprussiate (SNP) for 0, 24, 48, and 96 h. Relative water content (RWC) was increased by exogenous NO in YZ, but decreased in ZM. Except for glucose, fructose and fructans of degree of polymerization (DP) 3 in YZ, other soluble carbohydrates contents in the two wheat cultivars all increased to different degrees. The activities of FS (including sucrose: sucrose 1-fructosyltransferase (1-SST, EC: 2.4.1.99) and sucrose: fructan 6-fructosyltransferase (6-SFT, EC: 2.4.1.10)) were significantly higher than fructan: fructan 1-fructosyltransferase (1-FFT, EC: 2.4.1.100) in the seedlings of two wheat cultivars. The same phenomenon occurred to FBEs expression. In addition, sucrose content decreased while fructans content increased under low temperature, which was in accordance with the improved 1-FFT activity in ZM. Moreover, fructans content increased to a high level under high concentration of NO in ZM while kept at a constant low level in YZ. The expression levels of FBEs were universally higher in ZM than in YZ, which identified with the high frost resistance of the winter cultivar. It is concluded that exogenous NO treatment on wheat may be a good option to reduce chilling injury by regulating fructan accumulation in leaves. This is the first report owing that exogenous NO alleviated the negative effects of chilling stress by accumulating fructans in wheat.     

Anther Respiratory Activity and Chilling Resistance in Rice

The respiration of anthers in several rice cultivars differingin chilling resistance was analysed with Arrhenius plots. Theplots for respiration of mature anthers showed linearity ina temperature range between 7 and 35?C, whereas those of meioticanthers showed breaks at a temperature range between 18 and24?C. The break point in a given cultivar was nearly identicalto the temperature below which the meiotic anthers sufferedfrom chilling injury. The break in the Arrhenius plots indicatedthat the meiotic anthers were very susceptible to chilling.In addition, cultivars with relatively high chilling resistanceshowed lower critical temperatures for meiotic anther respirationthan those with relatively low resistance. Reciprocal F₁ hybridsbetween two cultivars with relatively high and low resistanceshowed almost the same respiratory parameters as those of theparent with high resistance, suggesting that the parameterswere inheritable as a dominant character. We postulate thatthere is a close correlation between the chilling sensitivityof anthers and the dependence of respiratory activity upon temperature,and that the critical temperature of meiotic anthers can betaken as an indicator of the degree of chilling resistance inrice plants. (Received March 17, 1984; Accepted July 25, 1984)     

Differential antioxidation activities in two alfalfa cultivars under chilling stress

To understand the adaptability of alfalfa (Medicago sativa L.) to chilling stress, we analyzed the antioxidative mechanism during seed germination. The germination rates of six alfalfa cultivars were studied comparatively at 10°C. Xinmu No. 1 and Northstar were selected as chilling stress-tolerant and stress-sensitive cultivars for further characterization. After chilling treatment, Xinmu No. 1 showed higher seedling growth than Northstar. Xinmu No. 1 exhibited low levels of hydrogen peroxide and lipid peroxidation compared with Northstar. In addition, shoots in Xinmu No. 1 treated with chilling showed higher activities of the superoxide dismutase, ascorbate peroxidase (APX), and catalase than those of Northstar, whereas Xinmu No. 1 showed higher APX activity in roots that Northstar. These results indicated that high antioxidation activity in Xinmu No. 1 under chilling stress is well associated with tolerance to chilling condition during germination.     

Postharvest chilling induces oxidative stress response in the dwarf tomato cultivar Micro-Tom

Oxidative stress is involved in the response of Lycopersicon esculentum fruits (cultivar Micro-Tom) to chilling. Changes in activated oxygen scavenging enzymes, superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2) were examined during ripening after postharvest chilling. Also, lipid peroxidation, respiration, and pigment contents were determined. These parameters were affected by chilling, especially the lycopene content and the respiration rate that showed a high value when the fruits were transferred to higher temperatures. CAT activity increased the day after the fruits were re-warmed, while the activity of GR was higher in the chilled than in the non-chilled green fruits. Lipid peroxidation was more evident at the 'pre-chilled' yellow and red fruits. APX and SOD were not affected by previous chilling in ripening fruits. These results indicate that oxidative stress is generated by conservation at 4°C. The antioxidant response of tomato fruit could be mediated by CAT and GR but not by SOD or APX. Moreover, CAT seemed to respond to the increase in the respiration rate.     

The electron partitioning between the cytochrome and alternative respiratory pathways during chilling recovery in two cultivars of maize differing in chilling sensitivity

Chilling effects on respiration during the recovery period were studied in two maize (Zea mays L.) cultivars differing in their tolerance to chilling: Penjalinan, a chilling-sensitive cultivar, and Z7, a chilling-tolerant cultivar. Both cultivars were exposed to 5 degrees C for 5 d, after which measurements were taken at 25 degrees C. Chlorophyll fluorescence analysis in dark-adapted leaves showed less damage in cv Z7 than in cv Penjalinan during recovery from the chilling treatment. Studies of the electron partitioning between the cytochrome and the alternative respiratory pathways during chilling recovery using the oxygen isotope fractionation technique showed that, although total leaf respiration was not affected by the chilling treatment in either of the two cultivars, electron partitioning to the alternative pathway was significantly increased in the more stressed chilling-sensitive cv Penjalinan, suggesting that increased activity of the alternative pathway is not related to the plant tolerance to chilling. These results suggest a possible role of the alternative pathway in plants under stress rather than specifically contributing to plant resistance to chilling.     

Response of photosynthetic capacity and antioxidative system of chloroplast in two wucai ( Brassica campestris L.) genotypes against chilling stress

Chilling stress during the growing season could cause a series of changes in wucai (Brassica campestris L.). WS-1 (chilling-tolerant genotype) and Ta2 (chilling-sensitive genotype) were sampled in present study to explore the chilling tolerance mechanisms. Our results indicated that photosynthetic parameters exhibited lower level in Ta2 than in WS-1 under chilling stress. The rapid chlorophyll fluorescence dynamics curve showed that chilling resulted in a greater inactivation of photosystem II reaction center in Ta2. Reactive oxygen species and malondialdehyde content of chloroplast in Ta2 were higher than WS-1. The ascorbate–glutathione cycle in chloroplast of WS-1 played a more crucial role than Ta2, which was confirmed by higher activities of antioxidant enzymes including Ascorbate peroxidase, Glutathione reductase, Monodehydroascorbate reductase and Dehydroascorbate reductase and higher content of AsA and GSH. In addition, the ultrastructure of chloroplasts in Ta2 was more severely damaged. After low temperature stress, the shape of starch granules in Ta2 changed from elliptical to round and the volume became larger than that of WS-1. The thylakoid structure of Ta2 also became dispersed from the original tight arrangement. Combined with our previous study under heat stress, WS-1 can tolerant both chilling stress and heat stress, which was partly due to a stable photosynthetic system and the higher active antioxidant system in plants, in comparison to Ta2.     

大豆种子吸胀冷害抗性与其质体膜脂不饱和度正相关

吸胀冷害是干种子在吸胀阶段遭受低温造成不萌发的现象,结果可能造成农作物损失严重。虽然吸胀过程中细胞膜的修复是关键事件,而且细胞膜在响应水分和温度胁迫中扮演重要角色,但是种子吸胀过程中膜变化的过程,特别是膜流动性变化过程研究较少。本文比较了吸胀冷害耐受型（LX）和敏感型（R5）两个大豆品种在吸胀冷害过程中膜脂不饱和度（double bond index, DBI）的变化,结果发现,LX和R5在常温（25℃）吸胀时变化趋势一致,质体膜脂DBI升高,质体外膜脂中磷脂酰甘油(phosphatidylglycerol, PG)分子DBI下降。LX和R5在低温（4℃）吸胀时DBI变化有很大差异,低温吸胀仅仅延缓了耐受型LX中质体膜脂DBI的升高,但是敏感性R5质体膜脂DBI不仅没有升高反而下降。用浓度33%的聚乙二醇 (polyethylene glycol, PEG)引发没有直接引起DBI变化,但是所引起的细微而显著的变化可能为萌发做好准备。PEG引发处理后的R5在吸胀冷害后第二和第三阶段质体膜脂DBI迅速增加,这个增加模式与LX的DBI增加相似。结果表明,吸胀冷害延缓或者阻滞了质体膜脂不饱和度的升高,大豆种子的吸胀冷害抗性与质体膜脂不饱和度正相关,提高质体膜质DBI可以提高吸胀冷害抗性。     

Phenological olive chilling requirements in Umbria (Italy) and Andalusia (Spain)

Phenological observations in olive tree (Olea europaea L.), on the first reproductive phase (budburst) and the winter chilling temperatures required for its onset, were analysed over a 4-year period (1998 - 2001). Research was carried out on two different cultivars growing in two Mediterranean olive-growing areas: 'Ascolana' in central Italy and 'Picudo' in southern Spain. The two main objectives of the study were: (1) to evaluate the different amounts of winter chilling, and their relationship with the budburst dates in outdoor olive plantations; and (2) to test the validity in the study areas of two chilling calculation methods, the Aron and the Utah method. Results show that for the Spanish cultivar the average chilling requirement of 997 h was approximately half of that recorded for the Italian cultivar (1848 h). Also, the year-to-year variability in chilling accumulation and the reproductive development date was observed to be higher in the Spanish area than in the Italian one, indicating a more consistent and predictable winter chilling response in the latter. As regards the validity of the methods, the Aron method seems to be more appropriate for use in warmer places, since it yielded better results in the Spanish site.