Probing the role of calmodulin in Al toxicity in maize

The role of calmodulin on Al toxicity was studied in two maize (Zea mays L.) inbred lines, Cat 100-6 (Al-tolerant) and S 1587-17 (Al-sensitive). Increasing levels of Al induced the release of malate at similar rate by roots of both genotypes, while the exudation of citrate, a stronger Al-binding compound, was 3.5 times higher in Cat 100-6 seedlings exposed to 16.2x10(-6) Al(3+) activity. The calmodulin inhibitor trifluoperazine significantly reduced the root growth in both genotypes, mimicking the main effect of Al. However, when Cat 100-6 and S 1587-17 seedlings were challenged with Al in conjunction with trifluoperazine, no further reduction in root growth or any other effect of Al toxicity was observed. The rate of Al-induced citrate exudation by both genotypes was not affected by treatment with trifluoperazine or calmidazolium, another calmodulin inhibitor. The Al(3+) interaction with cytoplasmic CaM was estimated using models for the binding of Al(3+) and Mg(2+) with CaM and physiological concentrations of citrate, CaM, InsP(3), ATP, ADP, Al(3+) and Mg(2+). In this simulation, Al(3+) associated with citrate and InsP(3), but not with CaM. We conclude that calmodulin is not relevant to the physiological processes leading to the Al tolerance in maize, nor is it a primary target for Al toxicity.     

Effect of anion channel antagonists and La3+ on citrate release, Al content and Al resistance in maize roots

The correlation between organic acid anion release and Al content was examined in two maize (Zea mays L.) inbred lines, Cat 100-6 (Al-resistant) and S 1587-17 (Al-sensitive) treated with anion channel antagonists and La3+, a cation channel blocker. In the intact roots of Al-resistant maize, the Al-induced excretion of citrate was inhibited by the anion channel antagonists niflumic acid, anthracene-9-carboxylic and ethacrinic acid. Citrate release in excised root apices was reduced by 60% in the presence of 15 microM niflumic acid, while the Al content increased by 42%. Nevertheless, Cat 100-6 accumulated less Al than S 1587-17 when the rate of citrate release was similar in both lines, indicating that other mechanisms of Al-resistance are operating in Cat 100-6. The presence of 60 microM La3+ did not change the rate of citrate release, but the Al content in excised root apices of Al-resistant plants was reduced by 70%. These results suggest that the Al distributed uniformly in the roots does not contribute to citrate release and possibly the activity of anion channels is correlated with the free activities of extracellular Al3+ close to anion channels.     

Gene expression profiling in maize roots under aluminum stress

To investigate the molecular mechanisms of Al toxicity, cross-species cDNA array approach was employed to identify expressed sequence tags (ESTs) regulated by Al stress in root tips of Al-tolerant maize (Zea mays) genotype Cat100-6 and Al-sensitive genotype S1587-17. Due to the high degree of conservation observed between sugarcane and maize, we have analyzed the expression profiling of maize genes using 2 304 sugarcane (ESTs) obtained from different libraries. We have identified 85 ESTs in Al stressed maize root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding previously identified proteins induced by Al stress, such as phenyl ammonia-lyase, chitinase, Bowman-Birk proteinase inhibitor, and wali7. In addition, several novel genes up-and downregulated by Al stress were identified in both genotypes.     

Somaclonal-variation-induced aluminum-sensitive mutant from an aluminum-inbred maize tolerant line

Somaclonal-variation-induced multiple mutations were observed in a progeny of the S1587 plant, regenerated from type I calli of the aluminum-tolerant inbred maize line Cat-100-6. After five generations of self-pollination, 14 progeny families of the S1587 somaclone were found to show aluminum toxicity symptoms with altered root tip morphology and reduced primary root growth. The most sensitive progeny, S1587-17, was crossed to the Cat-100-6 inbred line. The parental lines and the F1 were tested in nutrient solutions containing an aluminum activity gradient of 0–93 ⋅ 10^–6. The heterozygote behaves like the tolerant parent at aluminum activities up to 40 ⋅ 10^–6 and showed an intermediate phenotype at higher aluminum concentrations. Histological sections of aluminum-treated roots from tolerant and sensitive plants stained with hematoxylin, an aluminum marker, showed a progressive destruction of the root tip of the aluminum-sensitive genotype over time and indicated that tolerance in Cat-100-6 could be due to an aluminum exclusion mechanism. Segregation analysis of the F2 and backcross to the sensitive parent based on root morphology of plants subjected to an aluminum activity of 30 ⋅ 10^–6 showed the typical 3:1 and 1:1 tolerant:sensitive segregation ratios, respectively, indicating that tolerance in the Cat-100-6 inbred maize line is controlled by a single nuclear, semidominant gene, named Alm1. Received: 9 May 1996 / Revision received: 24 February 1997 / Accepted: 8 March 1997     

玉米幼苗抗氧化酶活性、脯氨酸含量变化及与其耐寒性的关系

研究了黄C（耐寒型）和Mo17（低温敏感型）两种耐寒性不同的玉米自交系幼苗在低温逆境（5 ℃,3 d）下,根、中胚轴和胚芽鞘3个部位过氧化氢酶（CAT）、过氧化物酶（POD）活性和脯氨酸（Pro）含量变化及与其耐寒性的关系.结果表明,低温期间,黄C的中胚轴、胚芽鞘相对生长率显著高于Mo17;黄C的根、中胚轴、胚芽鞘3个部位的CAT、POD活性和Pro含量变化率（其中,中胚轴分别为39.02%、-31.35%和86.86%）均高于Mo17（中胚轴变化率分别为1.86%、-48.67%和12.01%）;黄C 3个部位的质膜透性（中胚轴为22.05%）均低于Mo17（中胚轴为29.11%）.逐步回归分析表明黄C低温期间积累的干物质主要与Pro含量有关,而Mo17主要与POD活性有关,玉米幼苗中胚轴对低温反应最敏感,中胚轴Pro含量变化率与玉米耐寒性关系最密切.     

Organic acid exudation as an aluminum-tolerance mechanism in maize (Zea mays L.)

In this study, the role of root organic acid synthesis and exudation in the mechanism of aluminum tolerance was examined in Al-tolerant (South American 3) and Al-sensitive (Tuxpeño and South American 5) maize genotypes. In a growth solution containing 6 M Al³⁺, Tuxpeño and South American 5 were found to be two- and threefold more sensitive to Al than South American 3. Root organic acid content and organic acid exudation from the entire root system into the bulk solution were investigated via high-performance liquid chromatographic analysis while exudates collected separately from the root apex or a mature root region (using a dividedroot-chamber technique) were analyzed with a more-sensitive ion chromatography system. In both the Al-tolerant and Al-sensitive lines, Al treatment significantly increased the total root content of organic acids, which was likely the result of Al stress and not the cause of the observed differential Al tolerance. In the absence of Al, small amounts of citrate were exuded into the solution bathing the roots. Aluminum exposure triggered a stimulation of citrate release in the Al-tolerant but not in the Al-sensitive genotypes; this response was localized to the root apex of the Al-tolerant genotype. Additionally, Al exposure triggered the release of phosphate from the root apex of the Al-tolerant genotype. The same solution Al³⁺ activity that elicited the maximum difference in Al sensitivity between Al-tolerant and Al-sensitive genotypes also triggered maximal citrate release from the root apex of the Al-tolerant line. The significance of citrate as a potential detoxifier for aluminum is discussed. It is concluded that organic acid release by the root apex could be an important aspect of Al tolerance in maize.Abbreviations SA3 South American 3, an Al-tolerant maize cultivar - SA5 South American 5, an Al-sensitive maize cultivar The authors would like to express their appreciation to Drs. John Thompson, Ross Welch and Mr. Stephen Schaefer for their training and guidance in the use of the chromatography systems. This work was supported by a Swiss National Science Foundation Fellowship to Didier Pellet, and U.S. Department of Agriculture/National Research Initiative Competitive Grant 93-37100-8874 to Leon Kochian. We would also like to thank Drs. S. Pandey and E. Ceballos from the CIMMYT Regional office at CIAT Cali, Colombia for providing seed for the maize varieties and inbred line.     

水分胁迫对春播玉米苗期生长及其生理生化特性的影响

采用盆栽试验,以‘蠡玉18'玉米单交种为供试材料,设置充分供水(CK)、轻度水分胁迫(LS)、中度水分胁迫(MS)和重度水分胁迫(SS)4个水分处理水平,研究了水分胁迫对春播玉米苗期保护酶活性和生长的影响,以探讨土壤水分胁迫对玉米苗期生长发育及其生理过程的影响机制.结果表明:(1)随着水分胁迫程度的加剧,玉米幼苗的生物量显著下降,根冠比、根系活力和脯氨酸含量增加,且水分胁迫对玉米幼苗地上部生物量的抑制作用更大;可溶性蛋白含量差异不明显,MDA含量波动变化.(2)随着水分胁迫时间的延长,根冠比、根系活力和植株脯氨酸含量先升高后降低,可溶性蛋白含量呈先下降后升高的趋势;玉米幼苗叶片和根系MDA积累波动变化,而叶片MDA含量始终高于根系.(3)在水分胁迫初期,玉米叶片中CAT活性较SOD、POD响应更敏感;玉米苗期根系在中度水分胁迫下主要依赖CAT来降低氧化危害,而在重度水分胁迫下前期主要依赖CAT、后期通过CAT和POD的共同作用来降低氧化伤害;水分胁迫条件下,叶片和根系POD同步降低氧化伤害,而SOD和CAT在叶片和根系间存在互补作用.研究表明,在不同程度的水分胁迫条件下,玉米幼苗的生长受到一定程度的抑制,但其能够通过调节自身的保护酶活性和渗透调节物质含量来减轻干旱伤害,维持植株的正常生理代谢功能.     

Differential responses of antioxidant enzymes to aluminum toxicity in two rice (Oryza sativa L.) cultivars with marked presence and elevated activity of Fe SOD and enhanced activities of Mn SOD and catalase in aluminum tolerant cultivar

Seedlings of two Indica rice (Oryza sativa L.) cvs. HUR-105 and Vandana, differing in Al-tolerance were used to identify the key mechanisms involved in their differential behaviour towards Al toxicity. Cv. HUR-105 appeared to be Al sensitive by showing significant reduction (p ≤ 0.01) in root/shoot length, fresh weight, dry weight and water content in presence of 421 μM Al³⁺ in growth medium whereas cv. Vandana appeared to be fairly Al³⁺ tolerant. A conspicuous and significant reduction in dry weight of root and shoot was observed in Al sensitive cv. HUR-105 with 178 μM Al³⁺ treatment for 3 days. Al was readily taken up by the roots and transported to shoots in both the rice cultivars. Localization of absorbed Al was always greater in roots than in shoots. Our results of the production of reactive oxygen species (ROS) H₂O₂ and O₂ ^.? and activities of major antioxidant enzymes such as total superoxide dismutase (SOD), Cu/Zn SOD, Mn SOD, Fe SOD, catalase (CAT) and guaiacol peroxidase revealed Al induced higher oxidative stress, greater production of ROS and lesser capacity to scavenge ROS in cv. HUR-105 than Vandana. With Al treatment, higher oxidative stress was noted in shoots than in roots. Greatly enhanced activities of SOD (especially Fe and Mn SOD) and CAT in Al treated seedlings of cv. Vandana suggest the role of these enzymes in Al tolerance. Furthermore, a marked presence of Fe SOD in roots and shoots of the seedlings of Al tolerant cv. Vandana and its significant (p ≤ 0.01) increase in activity due to Al-treatment, appears to be the unique feature of this cultivar and indicates a vital role of Fe SOD in Al-tolerance in rice.     

Oxidative stress in herbicide-treated broad bean and maize plants

Treatments of broad bean and maize seedlings with fluometuron, atrazine or rimsulfuron affected some parameters of oxidative stress. Fluometuron significantly reduced activity of Hill reaction (PSII), chlorophyll a+b contents and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in leaves of both species and significantly increased contents of H₂O₂, lipid peroxides and carbonyl groups during the whole experiment. There were, moreover, significant inhibitions in activities of superoxide dismutase (SOD; E.C. 1.15.1.1), catalase (CAT; E.C. 1.11.1.6), ascorbate peroxidase (APX; E.C. 1.11.1.11) and guaiacol peroxidase (GPX; E.C. 1.11.1.7). Response to atrazine was, to some extent, similar to fluometuron throughout the entire experiment in broad bean and up mostly to the 12^th day of the experiment in maize. The herbicide effect was more pronounced in broad bean than maize. These results point to indicate an occurrence of oxidative stress in both species by fluometuron and only in broad bean by atrazine. The increase in H₂O₂ content accompanied with drop in activities of SOD, CAT and peroxidases indicates a decline in its detoxification rather than increase in its synthesis. On the contrary, rimsulfuron seemed to have no effect on most of the tested parameters although there were transient significant increases in H₂O₂, lipid peroxides and carbonyl groups as well as activities of SOD, CAT, APX and GPX. These findings, based on the recovery in oxidative stress, indicate that fluometuron is involved in oxidative stress generation in both species but atrazine only in broad bean while rismulfuron is not in both species.     

The ecotoxicological and interactive effects of chromium and aluminum on growth,oxidative damage and antioxidant enzymes on two barley genotypes differing in Al tolerance

The effects of single or combined stress of aluminum (Al) and chromium (Cr) on plant growth, root dehydrogenase, oxidative stress and antioxidative enzymes were studied using two barley genotypes differing in Al tolerance in a hydroponic experiment. Al or Cr stress decreased plant growth, lowered root dehydrogenase activity and caused oxidative damage, as characterized by increased MDA and H₂O₂ contents. Under Al or Cr stress, the activities of antioxidative enzymes, including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT), were dramatically increased in plant tissues. Gebeina, an Al-tolerant genotype, had less oxidative damage than Shang 70-119, an Al-sensitive genotype. The extent of oxidative damage induced by Cr varied with the pH of the culture solution, with lower pH values (4.0) being more severe than higher pH values (6.5). The combination of Cr and Al caused a further decrease in plant growth, a decrease in root dehydrogenase activity and an increase in MDA and H₂O₂ contents as well as the activities of antioxidative enzymes. There was also a marked difference between the two barley genotypes in the extent of increased antioxidative enzyme activity under the Cr and Al stresses.     

铜胁迫对狭叶香蒲生长及生理特性的影响

采用水培法,研究不同浓度Cu~(2+)胁迫对狭叶香蒲生长及生理特性的影响。结果表明:狭叶香蒲叶宽、株高的增长量及整株干物质累积量在较低浓度Cu~(2+)处理时均未受影响,此后随Cu~(2+)浓度升高显著下降。Cu~(2+)浓度为o～30 mg·L~(-1)时,叶绿素含量显著上升,此后随Cu~(2+)浓度增加其含量显著下降。在Cu~(2+)浓度0～5 mg·L~(-1)范围内根系活力显著上升,此后随Cu~(2+)浓度升高则大幅下降。根系和叶片SOD、POD、CAT、活性随Cu~(2+)浓度增加均呈先显著升高后显著降低的趋势,根系SOD、POD活性在30 mg·L~(-1)时出现最大值,叶片SOD、POD活性在55 mg·L~(-1)时出现最大值,根系和叶片CAT活性均在80 mg·L~(-1)时出现最大值;同一Cu~(2+)浓度下,根系SOD、POD、CAT活性明显高于叶片,说明根系比叶片对Cu~(2+)胁迫反应更敏感。从根系和叶片SOD/POD、SOD/CAT的比值变化上看出,在Cu~(2+)为1 mg·L~(-1)和5 mg·L~(-1)时起主要保护作用的酶是SOD、POD,后来CAT起到主要作用。MDA含量自Cu~(2+)浓度为30 mg·L~(-1)时开始持续上升。说明在Cu~(2+)为30～55 mg·L~(-1)时狭叶香蒲表现为积极的生理响应。     

Enhanced tolerance to sulfur dioxide and salt stress of transgenic Chinese cabbage plants expressing both superoxide dismutase and catalase in chloroplasts.

To explore the possibility of overcoming the highly phytotoxic effect of SO(2) and salt stress, we introduced the maize Cu/ZnSOD and/or CAT genes into chloroplasts of Chinese cabbage (Brassica campestris L. ssp. pekinensis cv. Tropical Pride) (referred to as SOD, CAT and SOD+CAT plants). SOD+CAT plants showed enhanced tolerance to 400 ppb SO(2), and visible damage was one-sixth that of wild-type (CK) plants. In addition, when SOD+CAT plants were exposed to a high salt treatment of 200 mM NaCl for 4 weeks, the photosynthetic activity of the plants decreased by only 6%, whereas that of CK plants decreased by 72%. SOD plants had higher total APX and GR activities than CK plants. As expected, SOD plants showed levels of protection from SO(2) and salt stress that were moderately improved compared to CK plants. However, CAT plants showed inhibition of APX activity and provided only limited improvements in plant stress tolerance. Moreover, SOD+CAT plants accumulated more K(+), Ca(2+) and Mg(2+) and less Na(+) in their leaves compared with those of CK plants. These results suggest that the expression of SOD and CAT simultaneously is suitable for the introduction of increased multiple stress protection.     

Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training

The purposes of this study were to determine whether exercise training induces increases in skeletal muscle antioxidant enzymes and to further characterize the relationship between oxidative capacity and antioxidant enzyme levels in skeletal muscle. Male Sprague-Dawley rats were exercise trained (ET) on a treadmill 2 h/day at 32 m/min (8% incline) 5 days/wk or were cage confined (sedentary control, S) for 12 wk. In both S and ET rats, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were directly correlated with the percentages of oxidative fibers in the six skeletal muscle samples studied. Muscles of ET rats had increased oxidative capacity and increased GPX activity compared with the same muscles of S rats. However, SOD activities were not different between ET and S rats, but CAT activities were lower in skeletal muscles of ET rats than in S rats. Exposure to 60 min of ischemia and 60 min of reperfusion (I/R) resulted in decreased GPX and increased CAT activities but had little or no effect on SOD activities in muscles from both S and ET rats. The I/R-induced increase in CAT activity was greater in muscles of ET than in muscles of S rats. Xanthine oxidase (XO), xanthine dehydrogenase (XD), and XO + XD activities after I/R were not related to muscle oxidative capacity and were similar in muscles of ET and S rats. It is concluded that although antioxidant enzyme activities are related to skeletal muscle oxidative capacity, the effects of exercise training on antioxidant enzymes in skeletal muscle cannot be predicted by measured changes in oxidative capacity.     

Al3+对大豆根边缘细胞程序性死亡诱导的生理生态作用

 设置不同的Al ³⁺浓度（0、25、50、100、200、400 μmol·L^-1）和培养时间 （12、24 h）,研究了边缘细胞活性和大豆（Glycine max）根中 过氧化氢酶（CAT）、过氧化物酶POD）、超氧化物歧化酶SOD）随Al ³⁺浓度及处理时间变化的规律,并通过Hoec hst333 42-PI双重荧光染色、 梯状DNA（即DNA ladder）分析和末端脱氧核糖核酸转移酶介导的dUTP切口末端标记（即TUNEL原位标记）检测,研究了Al ³⁺对大豆根边缘细胞 程序性死亡诱导的生理生态作用。结果表明,Al ³⁺胁迫能诱导边缘细胞的死亡,随着Al ³⁺浓度的升高和处理时间的延长,细胞死亡率增加。通 过Hoechst33342-PI双重荧光染色、DNA ladder分析和TUNEL原位标记,检测到Al ³⁺胁迫下发生程序性死亡的边缘细胞。其表现为：在 400μmol·L^-1 Al ³⁺诱导大豆根24 h时, 核酸电泳显示细胞DNA发生特异性降解并形成阶梯状电泳条带(DNA ladder),用TUNEL原位标记检测200 和400μmol·L^-1 Al ³⁺处理12 h后的大豆根 边缘细胞,发现DNA的3′-OH端被原位特异标记,二氨基联苯胺（DAB）显色后,细胞核为阳性或强 阳性。同时,高浓度Al ³⁺ （＞100μmol·L^-1）处理下,CAT、POD和S OD活性均有不同程度的下降,CAT和SOD的活性也随处理时间的延长而降低 。说明在Al ³⁺胁迫下边缘细胞的死亡可能是一种程序性死亡形式,高浓度Al ³⁺胁迫下,通过诱导活性氧在细胞体内的产生和累积而导致细胞凋 亡,此过程是其对逆境胁迫所作出的生理生态防御性应答方式之一。     

A patch-clamp study on the physiology of aluminum toxicity and aluminum tolerance in maize. Identification and characterization of Al(3+)-induced anion channels

The presence of Al(3+) in the rhizosphere induces citrate efflux from the root apex of the Al-tolerant maize (Zea mays) hybrid South American 3, consequently chelating and reducing the activity of toxic Al(3+) at the root surface. Because citrate is released from root apical cells as the deprotonated anion, we used the patch-clamp technique in protoplasts isolated from the terminal 5 mm of the root to study the plasma membrane ion transporters that could be involved in Al-tolerance and Al-toxicity responses. Acidification of the extracellular environment stimulated inward K(+) currents while inhibiting outward K(+) currents. Addition of extracellular Al(3+) inhibited the remaining K(+) outward currents, blocked the K(+) inward current, and caused the activation of an inward Cl(-) current (anion efflux). Studies with excised membrane patches revealed the existence of Al-dependent anion channels, which were highly selective for anions over cations. Our success in activating this channel with extracellular Al(3+) in membrane patches excised prior to any Al(3+) exposure indicates that the machinery required for Al(3+) activation of this channel, and consequently the whole root Al(3+) response, is localized to the root-cell plasma membrane. This Al(3+)-activated anion channel may also be permeable to organic acids, thus mediating the Al-tolerance response (i.e. Al-induced organic acid exudation) observed in intact maize root apices.     

Aluminium-responsive genes in sugarcane: identification and analysis of expression under oxidative stress

Suppression subtractive hybridization (SSH) technology was used to gain preliminary insights into gene expression induced by the phytotoxic aluminium species, Al(3+), in sugarcane roots. Roots of hydroponically-grown Saccharum spp. hybrid cv. N19 were exposed to 221 microM Al(3+) at pH 4.1 for 24 h, a regime shown to inhibit root elongation by 43%, relative to unchallenged roots. Database comparisons revealed that, of a subset of 50 cDNAs ostensibly up-regulated by the metal in the root tips, 14 possessed putative identities indicative of involvement in signalling events and the regulation of gene expression, while the majority (28) were of unknown function. All of the 50 cDNAs sequenced displayed significant similarity to uncharacterized plant expressed sequence tags (ESTs), approximately half (23) of which had been derived from other graminaceous crop species that had been subject to a variety of stresses. Analysis of the expression of 288 putative Al(3+)-inducible genic fragments indicated higher levels of expression under oxidative (1 mM diamide for 4 h) rather than Al(3+) stress. By deploying SSH, this study has provided an indication of the nature of genes expressed in sugarcane roots under Al(3+) stress. It is anticipated that the information obtained will guide further exploration of the potential for manipulation of the Al tolerance characteristics of the crop.