不同低温胁迫对烟草愈伤组织抗氰交替途径诱导和交替氧化酶表达影响的比较

比较了不同低温(14℃和4℃)胁迫对烟草(Nicotiana rusticaL．)愈伤组织抗氰交替途径诱导和交替氧化酶表达的影响。结果显示,不同低温胁迫处理能显著诱导烟草愈伤组织交替途径容量和实际运行的增加,且都呈现出基本相同的变化模式：在胁迫的初期(1—3d)持续增加,在3d时达到最高,而后下降到一个相对恒定的水平。但交替途径容量增加的幅度与温度下降的程度密切相关,而交替途径实际运行量的诱导程度在不同低温胁迫下的差异却很小。表明交替途径容量和实际运行对低温胁迫的响应是不同的。免疫印迹分析结果表明：低温胁迫明显诱导了交替氧化酶总蛋白的增加,且其随低温胁迫进程的变化与交替途径容量的变化基本一致;而对交替氧化酶单体与二聚体在低温胁迫下的含量变化检测结果则显示,烟草愈伤组织中交替氧化酶主要以二聚体形式存在,且这一存在形式并不随低温胁迫程度的加深而发生改变。两种形式的交替氧化酶蛋白含量都能被低温胁迫诱导增加,但其单体水平在两种不同的低温胁迫下并无明显差别,而4℃低温胁迫诱导的二聚体交替氧化酶蛋白含量明显高于14℃。表明不同程度低温对抗氰交替途径发生的不同影响主要是由于对交替氧化酶蛋白二聚体形式的不同诱导程度所致;而高活性的交替氧化酶单体形式则不因低温胁迫程度的加重而被明显诱导升高,使得抗氰交替途径的运行程度在两种不同的低温胁迫处理条件下无显著差异。     

不同低温胁迫对烟草愈伤组织抗氰交替途径诱导和交替氧化酶表达影响的比较

比较了不同低温（14℃和4℃）胁迫对烟草（Nicotiana rustica L.）愈伤组织抗氰交替途径诱导和交替氧化酶表达的影响。结果显示,不同低温胁迫处理能显著诱导烟草愈伤组织交替途径容量和实际运行的增加,且都呈现出基本相同的变化模式：在胁迫的初期（1～3 d）持续增加,在3 d时达到最高,而后下降到一个相对恒定的水平。但交替途径容量增加的幅度与温度下降的程度密切相关,而交替途径实际运行量的诱导程度在不同低温胁迫下的差异却很小。表明交替途径容量和实际运行对低温胁迫的响应是不同的。免疫印迹分析结果表明：低温胁迫明显诱导了交替氧化酶总蛋白的增加,且其随低温胁迫进程的变化与交替途径容量的变化基本一致;而对交替氧化酶单体与二聚体在低温胁迫下的含量变化检测结果则显示,烟草愈伤组织中交替氧化酶主要以二聚体形式存在,且这一存在形式并不随低温胁迫程度的加深而发生改变。两种形式的交替氧化酶蛋白含量都能被低温胁迫诱导增加,但其单体水平在两种不同的低温胁迫下并无明显差别,而4℃低温胁迫诱导的二聚体交替氧化酶蛋白含量明显高于14℃。表明不同程度低温对抗氰交替途径发生的不同影响主要是由于对交替氧化酶蛋白二聚体形式的不同诱导程度所致;而高活性的交替氧化酶单体形式则不因低温胁迫程度的加重而被明显诱导升高,使得抗氰交替途径的运行程度在两种不同的低温胁迫处理条件下无显著差异。     

盐胁迫下烟草愈伤组织内源活性氧及乙烯的产生与交替途径发生,？…

This paper reported firstly the dynamic changes of the alternative pathway and the cytochrome pathway in tobacco callus under different concentration of salt. The production rate of endogenous ethylene, the contents of active oxygen species (O2-., .OH and H2O2) and the activities of related enzyme (SOD and CAT) were also investigated simultaneously. The results indicated that the actual operation of the alternative pathway (rho Valt) increased gradually and reached the maxima at 0.75% salt concentration, then declined, while the capacity of the alternative pathway (Valt) didn't exhibit the same change pattern with rho Valt and the cytochrome pathway (rho' Vcyt) changed contrary to rho Valt. At the same time, H2O2 and O2-. accumulated rapidly and reached their peak value at about 0.5% and 0.75% salt concentration respectively, while the content of .OH increased consistently. The production rate of endogenous ethylene increased rapidly and reached its maxima at about 0.5% salt concentration. These data demonstrated that changes of Valt in tobacco callus were associated with a parallel production of H2O2 and endogenous ethylene and a opposite production of .OH, while rho Valt changed parally with the production of O2-.. On the basis of above results, it was deduced that the changes of the development and operation of the alternative pathway in tobacco callus under salt stress might be correlated with the production of endogenous ethylene and the active oxygen species. The possible participation of the alternative pathway in response to salt stress was also discussed.     

Effects of KCN and NaN3 Pretreatment on the Cyanide-Resistant Respiration in Tobacco Callus

The effects of KCN (0.5mmol/L) and NaN3 (0.01 mmol/L) pretreatment on the operation of the alternative pathway in subcultured tobacco (Nicotiana rustica L. cv. Gansu yellow flower) callus were analyzed. After treatment with KCN and NaN3 for 12 h, the total respiration rate (Vt) decreased by 12% and 17%, whereas oxygen consuption by the cytochrome pathway decreased by 22% and 28% respectively. The capacity of the alternative pathway (Valt) remained constant, while the activity of the alternative pathway (ρ· Valt ) inreased slightly. This changing pattern led to a declined contribution of the cytochrome pathway to the total respiration rate and an increased activity of the alternative pathway. Treatment with KCN for 24 h brought about a slight rise of oxygen consumption by the cytochrome pathway as compared with that in callus treated for 12 h, but the oxygen consumption was still lower than that in the untreated callus. Treatment with NaN3 for 24 h resulted in a profound decrease of the cytochrome pathway operation and a continuing increase of the alternative pathway operation. These data indicated that the enhanced operation of the alternative pathway played a compensatory role to the total respiration when the cytochrome pathway was partially inhibited in tobacco callus.     

烟草愈伤组织继代培养期间呼吸途径与活性氧水平动态变化

继代培养期间的烟草愈伤组织总呼吸速率分别在第11天和第19天出现两次跃升,同时抗氰呼吸的发生与运行逐渐加强,在两呼吸高峰之间达到极大值,随后则逐渐下降,但愈伤组织的电子传递仍以细胞色素途径为主．通过对愈伤组织衰老过程中活性氧代谢变化分析,发现抗氰呼吸发生与H     

小麦幼苗叶片抗氰呼吸对轻度水分胁迫的响应

小麦幼苗经－０．５ＭＰa聚乙二醇（ＰＥＧ－６０００）溶液暗中渗透迫０、６、１２、１８、２４、３０、３６、４２、４８h,叶片的总呼吸速率（Ｖt)呈现先上升后降低的趋势,交替途径呼吸也表现出相同的变化模式。水分胁迫初期（０－１２h),交替途径容量（Ｖalt)、实际运行活性（ρValt)及运行系数（ρ值）均上升,此后（１８－４８h）逐渐下降,水分胁迫也影响了呼吸电子流在２条呼吸途径中的分配比例,胁迫初期的０－１２h内,流经交替途径的电子流增多,而流向细胞色素主路的电子流减少,但随着胁迫时间的延长,交替途径的贡献降低,而细胞色素主路的贡献增加,说明小麦叶片的抗氰呼吸在水分胁迫初期被诱导增加,而随着胁迫进行的延长又表现为下降。     

Relationship Between Endogenous Ethylene and the Development and Operation of the Alternative Respiratory Pathway in Aged Potato Tuber Slices

The relationship between endogenous ethylene and the development and the operation of the alternative respiration pathway in aged potato (Solanum tuberosum L. ) tuber slices were investigated. During the 24 h aging period under 30 ℃, along with the great increase of the total respiratory rate (Vt) of the slices, the alternative pathway capacity (Valt) developed continuously, and the Valt/Vt values kept increasing as well. Both the alternative pathway activity (ρValt) and its contribution to Vt(ρValt/Vt) also increased gradually before 12 h of aging, but kept constant from 12 h to 24 h. The time course of the endogenous ethylene production of the aged slices was similar to the changing trends of Valt and Valt/Vt, but different from those of ρValt and ρValt/Vt values. ACC and Cu2 + treatment which stimulated ethylene production of the aged slices enhanced their Valt and Valt/ Vt values, Co2 + and Ag+ treatment resulted in a decreased effect. However, all the above treat ments of ethylene-related agents could not alter the continuously decreasing trend of the ρ value of the alternative pathway of the aged slices. And their effects on ρValt and ρValt/Vt values were only observed before 12 h of aging. These results suggested that the endogenous ethylene was essential to the development of the capacity of alternative pathway, but could only slightly influence the operation of the activity of the alternative pathway in aged potato tuber slices.     

盐逆境下转基因耐盐小麦与其受体呼吸途径的动态变化

以转基因耐盐品种８９１２２和其受体陇春１３号两种小麦为实验材料,研究科幼苗在不同盐浓度胁迫下呼吸途径动态变化。结果表明,８９１２２出现盐呼吸明显迟于其受体;两听Ｖａｌｔ与ρＶａｌｔ变化并不同步,且Ｖａｌｔ均受高盐浓度的抑制,但低盐浓度能诱导其受体的Ｖａｌｔ;两品种的ρＶａｌｔ与ρ′Ｖｃｙｔ彼此协同调节适应盐境,且ρ′Ｖｃｙｔ仍是盐胁迫过程中线粒体电子传递的主要途径。同时讨论了盐逆境下抗氰呼吸的一些     

The Relationship Between the Activity of Cyanide-resistant Respiration and the Expression of Alternative Oxidase in Different Organs of Mung Bean Seedlings

Twelve peptides, including eight conservative amino acid residues in the amino acid sequence of hydrophilic S helix of the alternative oxidase (AOX), were synthesized by solid-phase method. The polypeptide was coupled to αchymotrypsinogen, and the antibodies against this complex were obtained in rabbit. By using these antibodies, which were raised to immunoreact with total proteins of purified mitochondria from different organs of mung bean (Phaseolous radiatus L.) seedlings, it was found that there were two hybridizable AOX fractions in the mitochondria of mung bean seedlings. Their molecular weight was about 35 kD and 38 kD, respectively. Moreover, among the respiratory parameters obtained in hypocotyl, true leaf and cotyledon of mung bean seedlings true leaf had the highest total respiration (Vt), alternative pathway (AP) capacity(Valt) and the activity of AP (ρValt). Hypocotyl Vt and ρValt were the lowest, but its Vt was higher than that of the cotyledon. The activities of total and cyanide-resistant respiration were consistant with the analysis of Western blotting of AOX expression. The highest Vt and ρValt in true leaf were accompanied by two hybridizable polypeptides of AOX protein. The next was cotyledon Vt and ρValt with only one 38 kD hybridizable polypeptide of AOX protein. Hypocotyl Vt and ρValt were the lowest and its immunobloting band was similar to that of the cotyledon, but the expression amount of 38 kD protein was less than that of the cotyledon. The 35 kD AOX may make the main contribution to the true leaf ρValt.     

A Comparative Study of CN-Resistant Respiration in Different Cultures of Tobacco Callus

The callus of Nicotiana rustica cv Gansu yellow flower and N. tabacum cv willow leaf were cultured on ordinary subculture medium (M-1) and on regeneration medium (M-2), respectively. No differentiation was observed in Gansu yellow flower tobacco callus cultures grown on both M-1 and M-2 medium. The respiration of both cultures was partially resistant to cyanide and markedly inhibited by m-chlorobenzhydroxamic acid. The relative contributions of alternative and cytochrome pathway were 31% and 47% of the total respiration, respectively, in M-1 callus cultures. The relative O₂ uptake of the two pathways was not changed significantly in M-2 callus cultures. In subcultured M-1 callus cultures of Willow leaf tobacco, the respiration mediated via alternative pathway was about 29 to 38% of the total respiration, and the cytochrome pathway still was the major respiratory pathway. In M-2 callus cultures in which differentiation occurred, the relative contribution of alternative pathway increased to 41 to 47% of the total respiration, and the cytochrome pathway decreased considerably. These results suggested that the change of respiratory electron transport pathway was probably related to the differentiation of tobacco callus cultures.     

水杨酸对低温胁迫香蕉幼苗呼吸作用的影响

探讨了水杨酸 (salicylicacid ,SA)对低温胁迫香蕉幼苗叶片呼吸作用的影响。在常温下用 0 .5mmol/LSA水溶液处理香蕉幼苗 ,能明显提高香蕉幼苗的抗氰呼吸和细胞色素呼吸 ,增加总呼吸量 ,提高产热量 ;在随后 7℃低温胁迫与常温恢复期间SA预处理 ,能抑制总呼吸速率的下降 ,这种对总呼吸下降的抑制与此时细胞色素途径维持在较高的水平有关 ,而与抗氰呼吸无关。此时也未检测到SA预处理植株叶片产热量增加的现象     

H2O2和水杨酸对陈化马铃薯切片交替呼吸途径影响的比较

外源5.0 mmol/L H2O2和0.1 mmol/L 水杨酸(salicylic acid, SA)处理均可明显提高陈化24 h的马铃薯切片的交替呼吸途径容量(Valt)及其与总呼吸的比值(Valt/Vt).应用交替氧化酶的单克隆抗体进行Western杂交的结果表明,H2O2和SA处理均可明显提高陈化马铃薯切片中交替氧化酶的表达水平.用氧同位素分辨法研究,结果表明:H2O2处理对陈化马铃薯切片中交替呼吸途径的实际运行没有影响,而SA处理对交替呼吸途径的实际运行具有明显的促进作用.上述结果表明,H2O2和SA对植物组织交替呼吸途径的影响存在差异,二者均可促进交替氧化酶的表达从而诱导交替呼吸途径容量的发生,但H2O2不影响其实际运行,而SA还可同时诱导其实际运行.     

甘肃黄花烟草愈伤组织抗氰呼吸的研究

根据呼吸抑制剂试验和氧肟酸滴定法测定结果表明,甘肃黄花烟草愈伤组织呼吸中有明显的抗氰交替途径运行,平均占总呼吸的31%;但仍以细胞色素途径为主,平均占总呼吸的46%;还有23%不受 KCN 加 m-CLAM 抑制的未知剩余呼吸。改变培养基的激素成分和浓度,在不引起愈伤组织发生明显分化条件下,愈伤组织的生长和呼吸速率虽有不同,但抗氰交替途径和细胞色素途径对总呼吸的相对贡献程度和二者的变化趋势基本一致。     

Studies on the CN-resistant Respiration in Callus of Nicotiana rustica,Gansu Yellow Flower

In attempting to examine whether CN-resistant respiratory pathway is present in callus culture, we used tobacco callus cultures grown on different media. The M-1 medium contained tbe mineral and organic elements of MS medium and was supple,nented with 6-BA (0.5 mg/l) and 2,4-D (2 mg/l), and M-2 medium with 6-BA (2mg/l) and IAi (1 mg/l). No differentiation was observed in both of them. The respiration of M-1 callus was partly resistant to CN, and was markedly inhlbited by m-CLAM in the presence or absence of CN. Experiments of m-CLAM titration showed that the averages of relative contribution of alternative and cytochrome pathway in M-1 callus were 31% awl 46%of the total respiration respectively during the euliure period of 25 days. A same experiment was made on the M-2 callus. It was found that the pereeutages of relative contributions of the two electron transport pathways to the total respiration were approximately the same as those of the M-1 callus, although the respiratory rate was higher in M-2 callus. The above results showed that the bulk of respiratory electron flux was mediated by the eytoehrome pathway, although the alternative pathway was operative in callus of tobacco. The change of exogenous hormones added in the medium could not produee significant effects on the degree of relative contribution of two electron transport pathways under non-differentiation conditions.     

外源~1O_2 和·OH及其清除剂对烟草愈伤组织交替途径发生与运行的影响(英文)

外源1 O2 和·OH处理继代培养 1 4d的烟草愈伤组织 2 4h后 ,愈伤组织内的交替途径的实际运行显著上升 ,但对交替途径容量影响不大 ;而经·OH处理后的愈伤组织交替途径容量和实际运行均明显地受到抑制 ,但交替途径的实际运行对·OH更加敏感。活性氧产生系统中加入相关清除剂进行实验 ,得到与此相对应的结果 :1 O2 的清除剂His处理能明显地降低或抑制1 O2 所增加的交替途径实际运行量 ,但对交替途径容量则几乎均无影响 ,而用·OH的清除剂DMSO和MAN分别处理愈伤组织后 ,DMSO和MAN均能解除·OH对交替途径容量和实际运行的抑制。这些结果表明 ,1 O2 诱导烟草愈伤组织交替途径的实际运行 ,但对其容量的作用不大 ,而·OH则明显地抑制交替途径容量和实际运行。推测1 O2 和·OH对抗氰途径的影响可能是通过对AOX活性的调节     

Possible Participation of Active Oxygen Species in the Induction of Cyanide-Resistant Pathway at the Initial Senescence of Tobacco Callus

Changes in active oxygen species (AOS) and respiration, with special reference to the alternative pathway of respiration, were followed in callus of tobacco (Nicotiana rusticaL.) during its growth and senescence. Two peaks of the total respiration rate were observed: the first one appeared on day 11, and the second one on day 19 (in senescing callus). H₂O₂and O^– ₂contents increased gradually and reached the greatest values when callus senescence initiated (by day 11 and day 15, respectively), and then declined. The peaks of H₂O₂and O^– ₂before the onset of senescence coincided with the peaks of capacity V _altand activity V _altof alternative pathway, respectively. After the onset of senescence, ·OH accumulated abundantly and maintained at a relatively high level from then on, accompanied by the decrease in both V _altand V _alt. The conclusion is that these three active oxygen species operated coordinately to regulate the alternative pathway during growth and senescence of tobacco callus, as confirmed by the callus treatments with AOS scavengers, exogenous ·OH, and inhibitors of antioxidant enzymes. The possibility of AOS-induced alternative pathway respiration at the initial senescence of tobacco callus is discussed.     

Changes of the CN-resistant Respiration in Callus Cultures of Nicotiana tabacurn during the Period of Differentiation

The callus cultures obtained from unpollinated young ovaries of willow leaf tobacco were transplanted separately on subculture medium (M-l) and differentiation medium (M-2). They were called M-1 callus and M-2 callus respectively. Only meristematic cells and parenchymas could be observed in M-1 callus. The M-2 callus, however, was able to differentiation into vascular systems, embryoids and bud primordia. The respiration of M-1 callus exhibited CN-insensitive. The relative contributions of alternative pathway and eytochrome pathway to the total respiration were 29–38% and 44–51% respectively during the culture period of 25 days. It was obvious that the bulk of the respiratory electron flux was mediated by the eytochrome pathway. The greater part of the respiration in M-2 callus was insensitive to cyanide. The relative contributions of alternative pathway and cytochrome pathway to the total respiration were 41–47% and 29–32% respectively. These results suggested that the increase in the participation of the alternative pathway in total respiration was probablely related to the differentiation of the callus culture.     

葡萄休眠的自然诱导因子及其对休眠诱导期冬芽呼吸代谢的调控

以高需冷量葡萄品种‘夏黑’为试材,研究短日照、长日照和自然光照3个条件下,葡萄冬芽休眠的自然诱导因子及其对休眠诱导期冬芽呼吸代谢的调控机制.结果表明: 自然低温、短日照2个环境因素单独或共同作用均能诱导葡萄冬芽进行自然休眠.短日照在诱导葡萄冬芽进入自然休眠的过程中起主导作用,自然低温起辅助作用;温度相同条件下,日照时间越短对葡萄冬芽自然休眠的诱导作用越强.总呼吸速率达到峰值是葡萄冬芽休眠诱导期结束的标志.在自然休眠诱导期间,葡萄冬芽磷酸戊糖途径运行活性和容量占总呼吸的比例迅速上升,其中自然条件的葡萄冬芽分别由16.0%和20.1%上升至22.3%和26.0%.自然低温是诱导葡萄冬芽底物氧化水平上呼吸途径发生变化的主导因素,短日照起促进作用.在葡萄冬芽自然休眠诱导期间,交替途径运行活性和容量占总呼吸的比例迅速上升,其中自然条件葡萄冬芽分别由19.4%和27.3%上升至38.2%和46.8%.自然低温和短日照均可诱导葡萄冬芽电子传递链水平上呼吸途径发生变化.     

Comparative study of respiratory metabolism in three types of sugarbeet calli,a normal and two habituated ones (organogenic and non-organogenic)

The respiratory metabolism was studied in three types of sugarbeet (Beta vulgaris L. var. altissima) calli: a normal callus (N) and two fully habituated (auxin- and cytokinin-independent) calli, organogenic (HO) and non-organogenic (HNO). Except for the HO callus at day 14, the oxygen consumption rates of the habituated calli were always higher than that of the normal callus throughout the cycle of culture. The maximum activity of the cyanide-resistant pathway (alternative pathway) was much higher in the two habituated calli than in the normal one. By contrast, important differences were found in HNO and HO calli concerning the activity of the cytochrome pathway. In HNO cells, the high activity of this pathway was correlated with a high ATP level while the inverse situation was observed in HO cells. The physiological significance of these results is discussed.     

Changes in Respiratory Metabolism during Callus Growth and Adventitious Root Formation in Jerusalem Artichoke Tuber Tissues

The CN-resistant alternative oxidase pathway was examined inrelation to the formation of callus and adventitious roots ofJerusalem artichoke tuber tissues. During the early stage ofincubation at 28?C, respiration was significantly activatedand the O₂-uptake rate via the Cyt pathway increased 2.5–2.6times by the second day of culture regardless of the presenceof 2,4-D. However, further increases and preservation of highlevels of Cyt pathway activities were observed only in the callusformingtissues. The capacity of the alternative pathway also increased,but the actually operating fraction was very small (0–4%of total respiration). On the other hand, during the later stagesof culture in which adventitious roots emerged from the callus,activation of the alternative pathway was observed. These resultssuggested that the alternative pathway was not involved in callusgrowth but was related to root formation in callus tissues ofJerusalem artichoke tubers. (Received October 29, 1986; Accepted April 20, 1987)