Chilling stress and oxygen metabolizing enzymes in Zea mays and Zea diploperennis*

Abstract. The activities of five active-oxygen scavenging enzymes were compared for cold-lability and three were compared for chilling induction in two Zea genotypes of contrasting susceptibility to photoinhibition during chilling. Activities of superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GTR, EC 1.6.4.2) in leaf extracts from plants grown without chilling stress were assayed at 19°C and 5°C. Enzymes from the chilling-susceptible Z. Mays cv. LG11 had lower specific activities at 5°C than did enzymes from the chilling-tolerant Z. diploperennis, except for MDHAR where no significant differences were observed. The activities of SOD and APX from Z. diploperennis were double those of Z. mays at both assay temperatures. Monodehydroa-scrobate reductase and glutathione reductase activities in both species were reduced by 63–78% at a 5°C assay temperature. The dehydroascorbate reductase (DHAR) showed the greatest low-temperature lability losing 96% (Z. diploperennis) and 100% (Z. mays) of its activity at 5°C. To examine possible chilling-induced changes in levels of enzyme activity, plants of both Zea genotypes were transferred to growth chambers at 10°C at moderate light intensities. Glutathione reductase activity was found to increase within 24h in Z. diploperennis, but it decreased slightly in Z. mays. MDHAR activity decreased by 50% in Z. diploperennis but showed only a transient increase in activity in Z. mays.     

Genotype-specific differences in chilling tolerance of maize in relation to chilling-induced changes in water status and abscisic acid accumulation

Four inbred maize lines differing in chilling tolerance were used to study changes in water status and abscisic acid (ABA) levels before, during and after a chilling period. Seedlings were raised in fertilized soil at 24/22°C (day/night), 70% relative humidity. and a 12-h photoperiod with 200 μmol m⁻² s⁻¹ from fluorescent tubes. At an age of 2 weeks the plants were conditioned at 14/12°C for 4 days and then chilled for 5 days at 5/3°C. The other conditions (relative humidity, quantum flux, photoperiod) were unchanged. After the chilling period the plants were transferred to the original conditions for recovery. The third leaves were used to study changes in leaf necrosis, ion efflux, transpiration, water status and ABA accumulation. Pronounced differences in chilling tolerance between the 4 lines as estimated by necrotic leaf areas, ion efflux and whole plant survival were observed. Conditioning significantly increased tolerance against chilling at 5/3°C in all genotypes. The genotypes with low chilling tolerance had lower water and osmotic potentials than the more tolerant genotypes during a chilling period at 5/3°C. These differences were related to higher transpiration rates and lower diffusive resistance values of the more susceptible lines. During chilling stress at 5/3°C ABA levels were quadrupled. Only a small rise was measurable during conditioning at 14/12°C. However, conditioning enhanced the rise of ABA during subsequent chilling. ABA accumulation in the two lines with a higher chilling tolerance was triggered at a higher leaf water potential and reached higher levels than in the less tolerant lines. We conclude that chilling tolerance in maize is related to the ability for fast and pronounced formation of ABA as a protective agent against chilling injury.     

Depolymerization of cortical microtubules is not a primary cause of chilling injury in corn (Zea mays L. cv Black Mexican Sweet) suspension culture cells

Cold-induced depolymerization of cortical microtubules were examined in suspension culture cells of corn (Zea mays L. cv Black Mexican Sweet) at various stages of chilling. In an attempt to determine whether microtubule depolymerization contributes to chilling injury, experiments were carried out with and without abscisic acid (ABA) pretreatment, since ABA reduces the severity of chilling injury in these cells. Microtubule depolymerization was detectable after 1 h at 4°C and became more extensive as the chilling was prolonged. There was little chilling injury after 1 d at 4°C in either ABA-treated or non-ABA-treated cells. After 3 d at 4°C, there was about 26% injury for ABA-treated and 40% injury for non-ABA-treated cells, as evaluated by 2,3,5-triphenyl-tetrazolium chloride reduction and by regrowth. After 1d at 4°C, less than 10% of cells retained full arrays of microtubules in both ABA-treated and non-ABA-treated cells, the remainder having either partial arrays or no microtubules. After 3d at 4°C, about 90% of cells showed complete or almost complete depolymerization of microtubules in both ABA-treated and non-ABA-treated cells. ABA did not stabilize the cortical microtubules against cold-induced depolymerization. In about 66% of ABA-treated cells and 57% of non-ABA-treated cells that had been held at 4°C for 3d, repolymerization of cortical microtubules occurred after 3h at 28°C. These results argue against the hypothesis that depolymerization of cortical microtubules is a primary cause of chilling injury.     

Monitoring chilling injury: A comparison of chlorophyll fluorescence measurements, post-chilling growth and visible symptoms of injury in Zea mays

Changes in chlorophyll fluorescence emission from maize ( Zea mays L. cv. Northern Belle) seedlings chilled at 1.5°C in the dark for 3–30 h were compared with the ability of plants to resume growth in the immediate post-chilling period and with the development of visible symptoms of injury to the leaves. During chilling, the maximal rate of increase of the induced chlorophyll fluorescence rise. F_R, was measured on secondary leaf tissue. F_R decreased exponentially, at approximately the same rate in plants grown and chilled in hydroponic pots, in leaves detached from similar plants and in plants that were removed from the hydroponic pots and laid on wet filter paper adjacent to the detached leaves. The half-fall time for F_R in the 3 treatments was 7.8 ± 1.3 h, 8.6 ± 0.6 h and 8.8 ± 1.0 h, respectively. Following seedling removal from 1.5°C and return to 25/15°C, relative growth rates were determined from daily measurements of plant fresh weight gain. Compared with non-chilled seedlings, plants chilled for 3 h and longer showed depressed rates of growth. Inhibition of growth in the immediate post-chilling period (0–27 h) was linearly related to the duration of the chilling period and had a high positive correlation with the decrease in chlorophyll fluorescence (linearly related to log F_R) sustained during the chilling exposure. Visible symptoms of chilling injury developed during the post-chilling period on seedlings chilled for longer than 3 h. The decrease in log F_R during chilling was also linearly correlated with the severity of visual symptoms of chilling injury expressed in the post-chilling period. It is concluded that the extent of chilling injury in maize can be rapidly and non-destructively assessed from measurements of chlorophyll fluorescence.     

Low growth temperature-induced changes to pigment composition and photosynthesis in Zea mays genotypes differing in chilling sensitivity

The effects on pigment composition and photosynthesis of low temperature during growth were examined in the third leaf of three chilling-tolerant and three chilling-sensitive genotypes of Zea mays L. The plants were grown under a controlled environment at 24 or 14 °C at a photon flux density (PFD) of 200 or 600 μ mol m^–2 s^–1. At 24 °C, the two classes of genotypes showed little differences in their photosynthetic activity and their composition of pigments. At 14 °C, photosynthetic activity was considerably reduced but the chilling-tolerant genotypes displayed higher photosynthetic rates than the chilling-sensitive ones. Plants grown at 14 °C showed a reduced chlorophyll (Chl) a + b content and a reduced Chl a / b ratio but an increased ratio of total carotenoids to Chl a + b . These changes in pigment composition in plants grown at low temperature were generally more pronounced in the chilling-sensitive genotypes than in the tolerant ones, particularly at high PFD. Furthermore, at 14 °C, all the genotypes showed increased ratios of lutein, neoxanthin and xanthophyll-cycle carotenoids to Chl a + b but a reduced ratio of β -carotene to Chl a + b , especially at high PFD. At 14 °C, the chilling-tolerant genotypes, when compared with the sensitive ones, were characterized by higher contents of β -carotene and neoxanthin, a lower content of xanthophyll-cycle carotenoids, a lower ratio of xanthophylls to β -carotene, and less of their xanthophyll-cycle carotenoid pool in the form of zeaxanthin. These differences between the two classes of genotypes were more pronounced at high PFD than at low PFD. The results are discussed in terms of the relationship that may exist in maize between pigment composition and the capacity to form an efficient photosynthetic apparatus at low growth temperature.     

Environmental effects on the early stages of tassel morphogenesis in maize (Zea mays L.)

The effects of various environmental conditions on the initiation of tassel branches (NTB) and spikelet‐pairs (NSP) were examined in the stress‐sensitive maize inbred F53. Chilling induced the most important effect, with a dramatic decrease in both NTB and NSP, provided it was applied at the end of the vegetative phase and start of the floral transition phase. The primary cause of chilling‐induced abortion of the tassel branches could be oxidative stress in the leaves, since lowering light irradiance during chilling greatly reduced the effect of cold. The comparison of inbreds F53 and F2 revealed that both genotypes exhibited a similar period of cold sensitivity at the floral transition phase, although F2 was considered from field observations as a stress‐insensitive genotype (at least for tassel development). However, our results also showed a chilling acclimation response in inbred F2 but not in inbred F53. The similarities with the work by Lejeune & Bernier (1996 Plant, Cell and Environment 19, 217–224.) concerning the effect of chilling on ear initiation in the sensitive inbred, B22, are emphasized.     

玉米种子萌发能力和耐脱水能力的形成

以玉米品种“粤单9117”为材料,研究了种子发育过程中萌发能力和耐脱水能力的获得。玉米种子的生理成熟期约为43DAP（授粉后天数）。胚萌发能力的获得是在14－21DAP、耐脱水能力的获得出现在25－28DAP。胚的耐脱水能力在28DAP后仍不断得到加强。耐脱水能力的获得与细胞膜的发育及受保护的程度密切相关。脱水有利于不同发育时期的胚和种子的萌发。     

玉米(Zea mays L.)叶脉发育的研究

玉米的叶脉在单子叶植物中有一定的代表性,叶脉由四级组成,粗细不同的一、二、三级叶脉均从叶基向叶尖方向延伸,属叶片的纵向叶脉,四级脉横向与一、二、三级叶脉连接,是横向的叶脉,各级叶脉有各自的形成方式,由于它们有规律的分布,从而构成了叶片的输导网络,各级叶脉的发生和发育与叫片的生长有直接的关系。     

Salt tolerance of maize (Zea mays L.): the role of sodium exclusion

The influence of NaCl and Na₂SO₄ on growth of two maize cultivars (Zea mays cv. Pioneer 3906 and cv. Across 8023) differing in Na⁺ uptake was investigated in two green-house experiments. Na⁺ treatment with different accompanying anions (Cl^?/SO₄^2?) showed that ion toxicity was caused by Na⁺. While shoot growth of the two cultivars was markedly affected by salt in comparison to the control during the first 2–3 weeks, there were only slight differences between the cultivars. The shoot Ca²⁺ concentration was reduced in both cultivars, and the youngest leaves contained an even lower concentration compared with the rest of the shoot. During this first phase, Across 8023 tended to have higher concentrations of Ca²⁺ than Pioneer 3906. The Na⁺-excluding cultivar Pioneer 3906 showed continuous, although reduced, growth compared with the control, while the Na⁺ concentration in the shoot decreased until flowering. Cultivar Across 8023 accumulated Na⁺ until flowering: the reduction in the growth of stressed plants was greater than that for Pioneer 3906. Leaves of cultivar Across 8023 showed clear toxic symptoms, while those of the more salt-tolerant cultivar Pioneer 3906 did not. It is concluded that Na⁺ exclusion contributes to the salt tolerance of maize.     

萌发玉米种子脱水耐性与胚根细胞超微结构的变化

玉米种子脱水试验表明,25℃下萌发24h种子脱水耐性开始丧失,丧失50％和100％的时间分别为33h、58h。萌发过程中随着吸胀时间增加,玉米种子脱水耐性逐步丧失。显微观察显示,种子吸胀过程中,胚根细胞的贮藏物质逐步减少,线粒体等细胞器的分化程度则不断提高,尤其是脂类物质的分解程度与脱水耐性变化的关系似乎更明显。     

Calcium-induced sperm fusion in Zea mays L.

 In this study, we examined morphological changes of isolated maize (Zea mays L.) sperm cells in the presence of Brewbaker and Kwack salts (BKS) or the individual components of BKS using light, transmission electron and scanning electron microscopy. Freshly isolated sperms are 7.5 μm in diameter. Treatment with BKS for 5 h resulted in large cells with a diameter up to 41 μm. Staining of sperm nuclei with 4′, 6-diamidino-2-phenylindole (DAPI) revealed two or more nuclei in a single cell, suggesting that BKS induces cell fusion. Treatment with each BKS component showed that cell fusion occurs only in the presence of calcium nitrate. Use of several calcium salts showed the same results, suggesting that the calcium ion, alone, is responsible for the observed cell fusion. Further studies were conducted to examine the relationship between calcium distribution and sperm location in pollen tubes using chlorotetracycline and DAPI. Growing maize pollen tubes exhibited a high membrane calcium region within 20–50 μm from the tip. The Sperms are found no closer than 90 μm to the tip of the tube, suggesting that sperms are located in a low calcium region prior to being released to the degenerating synergid. Received: 12 August 1996 / Revision accepted: 6 December 1996     

玉蜀黍轴及苞叶对凝血作用的影响

采用体外血浆复钙时间法,以维生素k1作为促凝血和灯盏花素作为抗凝血作用阳性对照,对玉蜀黍轴及苞叶石油醚、乙酸乙酯和正丁醇提取物及玉蜀黍苞叶甲醇总浸膏对体外血浆复钙时间的影响进行测定。结果显示玉蜀黍轴正丁醇及苞叶乙酸乙酯提取物,均可显著缩短体外血浆复钙时间(P<0.001);玉蜀黍苞叶石油醚提取物可显著延长体外血浆复钙时间(P<0.001)。提示玉蜀黍轴正丁醇及苞叶乙酸乙酯提取物具有较好的促凝血活性,玉蜀黍苞叶石油醚提取物具有较好的抗凝作用。     

不同基因型玉米间作的群体质量

采用大田试验,研究了不同基因型玉米间作的群体质量特征.结果表明,HF9‖XD20间作,植株中部叶片平均叶龄延长,而对下部和上部叶片影响不大;ZD958‖LD981间作,ZD958植株下部叶片平均叶龄延长,而中、上部叶片则缩短,LD981植株下、中、上部叶片平均叶龄均有所延长.吐丝前,群体叶面积指数(LAI)单间作无明显差异,吐丝后,HF9和LD981的LAI分别大于和显著大于单作群体,而ZD958和XD20则分别小于和显著小于单作群体.紧凑型品种和半紧凑型品种间作增加了群体透光率,吐丝后10d,4个品种棒三叶叶色值(SPADR)均有所增加,并且除ZD958外,其余3个品种棒三叶净光合速率均有所增加,其中LD981增加显著.间作对吐丝以前的群体干物质积累量影响不大,吐丝后,半紧凑型品种(HF9和LD981)的干物质积累量增加,其中LD981增加显著,而紧凑型品种(XD20和ZD958)的干物质积累量减少,其中ZD958显著减少;间作还提高了收获指数,并且两种间作群体的土地当量比(LER)均大于1.结果提示,紧凑型与半紧凑型玉米品种的间作可以提高群体质量,延长叶片功能期,提高光合效率,增加籽粒产量.     

Plant Regeneration from Protoplasts of Corn(Zea mays L.)

Maize embryogenic calli induced from pollen were subcultured for one and one half years on N, basic medium supplemented with 2 mg/1 kinetin, 1 mg/l 6-benzyl-aminopurine, 0.3 mg/l 2,4-D, 500 mg/l casein hydrolysate and 250 mg/l glutamine. These embryogenic calli were used for protoplast isolation. Protoplasts were cultured on Z2 medium (Table 1) which is composed of rice protoplast culture basic medium 1 supplemented with 0.2 mg/l kinetin, 0.1 mg/l 6-benzyl-aminopurine, 0.5 mg/l 2,4-D, 200 mg/l casein hydrolysate, 100 mg/l glutamine and 2% coconut milk. The first division of regenerated cell occurred after 4-6 days in culture. After 3 weeks later, small calli could be seen with naked eyes. At this moment, addition of the same Z2 medium with decreased osmoticum twice for the protoplast culture is necessary. Regenerated calli, 2–4 mm in diameter, were transferred in succession on differentiation medium Z3 and Z4 for organogenesis. Embryogenesis and plant regeneration could occur simultaneously on Z4 differentiation medium. It seems that except the cultural conditions genotype and using of embryogenic materials are the two key factors for plant regeneration of maize protoplast and the former may be the critical one.     

Leaf photosynthetic parameters of two maize (Zea mays) cultivars in response to various patterns of chilling temperatures and photon flux densities

Chilling‐induced photosynthetic impairment was examined in leaves of maize (Zea mays L.) seedlings of two cultivars, one adapted to western Europe and one adapted to Mexican highlands. Three experiments were performed in a controlled environment. The effects of chilling night temperatures, of chilling at high light intensity and of variable chilling day temperatures on photosynthetic parameters, were evaluated. Chilling in the dark period resulted in stomatal limitation of net photosynthesis. Chilling at moderate to high light intensities caused chilling‐dependent photoinhibition of CO₂ uptake. Photobleached maize leaves did not resume normal photosynthetic function. Maize cv. Batan 8686 from the highlands of Mexico was less susceptible to photosynthetic damage than maize cv. Bastion adapted for cultivation in W. Europe, when exposed to chilling night temperatures, or to mild chilling photoinhibitory conditions.     

Light-dependent Proton Excretion of Wheat (Triticum aestivum L.) and Maize (Zea mays L.) Roots

We investigated the change of root net proton excretion of seedlings of Triticum aestivum L. and Zea mays L. with daily variation of illumination using a multi-channel pH-stat system. We found an increase of net proton excretion during darkness and a drop after the beginning of illumination. Inhibition of carotenoid biosynthesis by norflurazone and photooxidation of chlorophylls did not change the periodicity or its induction. The induction of diurnal periodicity was possible with blue, green and red light. After induction the oscillation of net proton excretion continued for at least two cycles under constant light. We conclude that net H⁺ excretion of wheat and maize roots may be regulated by an endogenous clock or by a signal from the leaves. The nature of such a hypothetical signal remains unknown.     

Glykosidasen und saure Phosphatase in isolierten Vakuolen aus Wurzelspitzen von Zea mays L

Aus Maiswurzelspitzen werden mechanisch meristematische Vakuolen isoliert, die nur geringfügig kontaminiert sind. Mit optimierten Testverfahren werden drei Glykosidasen und die saure Phosphatase untersucht. Die β-Glucosidase- und die β-Galaktosidase-Aktivitäten sind im zur Vakuolenreinigung benutzten Gradienten nahezu völlig übereinstimmend verteilt (ein Enzym oder identische Kompartimentierung beider Enzyme?). Nur minimale Aktivitäten (Kontamination?) liegen in der Vakuolenfraktion vor, hohe dagegen im Gradientensediment, was auf eine vorwiegende Assoziation dieser Enzyme mit der Zellwand und/oder daran haftenden Plasmalemmaresten hinweist. In der Vakuolenfraktion sind die α-Glucosidase und die saure Phosphatase angereichert. Diese Enzyme sind also zumindest partiell in der Vakuole lokalisiert. Die α-Glucosidase ist zusätzlich (ähnlich den β-Glykosidasen) im Gradientensediment angereichert. Die α-Glucosidase- und β-Glykosidase-Aktivitäten gehen wohl auf verschiedene Enzyme zurück. Die Verwendung der sauren Phosphatase als Vakuolenmarker ist zweifelhaft und die Notwendigkeit eines allgemeinen, spezifischen Vakuolenmarkers offensichtlich. Die Untersuchungen wurden im Labor von Professor Dr. R. Wiermann an der Universität Münster durchgeführt und von der Deutschen Forschungsgemeinschaft unterstützt. Frau Dr. U. Dohrmann danke ich für die kritische Durchsicht des Manuskriptes und Frau M. Boldt für die hervorragende technische Assistenz bei der Optimierung der enzymatischen Testverfahren.