棉花叶片衰老过程中激素和膜脂过氧化的关系

以陆地棉品种辽棉9号的去根幼苗为材料,对其进行暗诱导衰老培养.在培养液中分别加入6-BA、ABA、GSH、H2O2、CaCl2、A23187 和A23187 CaCl2,测定在不同培养条件下棉花去根幼苗叶片内源激素、SOD酶活性和MDA含量的变化.结果表明:棉花叶片衰老表现为细胞分裂素含量的下降和ABA含量的上升.6-BA、GSH和钙离子均延缓叶片的衰老,ABA和H2O2促进叶片的衰老.     

大豆叶片衰老过程中质膜蛋白激酶自磷酸化状态和催化活性的变化

以大豆幼苗初生叶为材料研究了衰老过程中质膜蛋白激酶自磷酸化状态和催化活性的变化。结果发现质膜上一个57kD的蛋白激酶分子上有多个自磷酸化位点,而且自磷酸化反应能提高该酶催化组蛋白H1磷酸化的激酶活力。进一步的研究表明诱导衰老处理造成的57kD蛋白激酶自磷酸化状态的变化,可能对调节它在衰老过程中催化活性的变化起重要作用;而外源6-BA预处理则能够维持57kD蛋白激酶体内高自磷酸化状态,保持该激酶在衰老过程中的催化活力。对衰老和6-BA处理过程中质膜上39和47kD蛋白激酶自磷酸化状态变化的研究表明,这两种激酶可能参与大豆叶片对6-BA刺激信号的传导和/或应答反应过程。     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

曼陀罗细胞中6-BA结合蛋白与硝酸还原酶活力的关系

曼陀罗单倍体细胞,在无水层情况下,经紫外线诱变,在氯酸盐培养基上筛选得到一株缺少硝酸还原酶活力的突变细胞株。经3年培养证明突变性状稳定。对它进行生理生化分析,看到它核内细胞分裂素结合蛋白减少。结合蛋白与细胞分裂素结合后能够促进核内 RNA 聚合酶活力,加速基因转绿。在正常细胞中,细胞分裂素能够促进硝酸还原酶诱导活力,而在这些缺少核内细胞分裂素结合蛋白的突变细胞中,外源细胞分裂素对酶活力的诱导没有作用。从而猜测,在突变细胞中,由于激素不能充分与结合蛋白结合,降低了细胞中转绿的总水平,可能是硝酸还原酶活力提不高的原因。本文还讨论了激素与受体结合后在调控基因表达上的可能途径。     

Reduction of Cytokinin Binding-protein in a Nitrate Reductase Deficient Mutant Cell Line of Datura inoxia

Suspension culture cells initiated from haploid Datura inoxia seedlings were transferred on a paper and were treated with UV. The nitrate reductase (NR) deficient mutants were isolated by selection for chlorate resistance. The NR activity could not be recovered, even though the mutants were transferred into the medium without selective pressure for three years. Isoelectrofocusing gel showed that the gene of NR was not destroyed by the treatment of UV. The mutant cells were defective in the cytokinin binding protein. The cytokinin binding-protein was isolated from wheat seedlings with the aid of 6BA immobilized on the epoxy-sepharose colunm. An addition of binding-protein, together with 6BA, to the medium for synthesis of RNA in vitro brough about an activation of RNA-polymerase. In wild type cells the NR activity was accelerated by the addition of cytokinin to the culture medium. In contrast, cytokinin was of no effect on the synthesis of NR in mutant cells. It is, therefore, suggested that the effect of cytokinin on the RNA synthesis and NR formation was regulated by the content of cytokinin binding-protein in Datura inoxia mutant cells.     

Reduction of paraquat toxicity in maize leaves by benzyladenine

The protective effect of a cytokinin benzyladenine (BA), against toxicity of paraquat (PQ), a widely used herbicide and a well-known oxidative stress inducer, was investigated in the leaves of maize. Maize leaves have been pretreated with BA at concentrations of 1, 10 and 100 microM and afterwards treated with PQ. At all concentrations tested, BA retarded PQ-induced decreases in chlorophyll, carotenoid and ascorbic acid contents. Pretreatment with 10 and 100 microM of BA significantly increased superoxide dismutase (SOD) activity after 8 h of PQ treatment but there was no significant change in SOD activity in the leaves pretreated with BA at 12 and 24 h. However, peroxidase activity significantly increased in 100 microM of BA pretreated leaves. Results indicate that pretreatment with BA reduce PQ toxicity and BA-treated plants might become more tolerant against oxidative stress.     

外源MeJA对高温胁迫下半夏抗氧化系统和胁迫基因的影响

研究叶面喷施外源MeJA对高温胁迫下半夏的超氧化物歧化酶(SOD)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)活性的影响;对脯氨酸(Pro)、丙二醛(MDA)、可溶性糖含量及有效成分含量的影响;对小分子热激蛋白(sHSP)等胁迫相关基因表达的影响。将生长状况一致的半夏植株在40℃高温下进行胁迫处理,实验组喷施50μmol·L^-1的外源MeJA溶液,空白组喷施等量的水,分别在处理2、6、12、24、48 h时进行取样,对样品进行指标测定。结果发现,在40℃高温下,喷施一定浓度的外源MeJA可以提高半夏叶片的SOD、POD、APX活性,降低MDA质量分数,增加脯氨酸及可溶性糖含量;对半夏有机酸含量无明显影响。2个细胞质型小分子热激蛋白和GRRBP蛋白,在MeJA处理后表达量显著增加。由此得出结论:喷施外源MeJA可以增强半夏中部分抗氧化酶的活性,保护半夏的细胞膜,增强细胞的渗透调节能力,且外源MeJA可能增加半夏高温胁迫响应基因的表达。     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Effects of methyl jasmonate (MeJA) on the dark-induced senescence in oat (Avena sativa L.) leaf segments

To investigate the relationship between methyl jasmonate (MeJA) and ethylene in leaf senescence, we studied the effects of MeJA on ethylene production and ethylene biosynthetic enzyme activities in oat(Avena sativa L.) leaf segments incubated in darkness. MeJA promoted dark-induced senescence judged from the contents of chlorophyll and protein, and increased ethylene production 6 times of the control. MeJA also increased the activities of ethylene biosynthetic enzymes, 1-aminocyclopropane carboxylic acid (ACC) synthase and ACC oxidase as compared to control. In MeJA-treated leaf segments, ACC synthase activity reached its maximum level at 24 h of incubation and ACC oxidase activity peaked at 6 h of incubation. Aminoethoxyvinylglycine (AVG) and Co²⁺, inhibitors of ACC synthase and ACC oxidase respectively, reduced MeJA-induced ethylene production. They also delayed leaf senescence that was promoted by the treatment of MeJA. From these results, we can suggest that MeJA increased the activities of ACC synthase and ACC oxidase, these increased activities lead to increase in ethylene production and this increased ethylene production might promote dark-induced leaf senescence.     

Nitric oxide counteracts the senescence of detached rice leaves induced by dehydration and polyethylene glycol but not by sorbitol

In the present study, we evaluated the protective effect of nitric oxide(NO) against senescence of rice leaves enhanced by water deficit. Dehydration(DH), polyethylene glycol (PEG) and sorbitol (ST) treatments were used toinducewater deficit. Senescence of rice leaves was determined by the decrease ofprotein content. NO donors[N-tert-butyl--phenylnitrone (PBN), sodiumnitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and ascorbic acid +NaNO₂] were effective in inhibiting senescence of dehydrated andPEG-treated rice leaves, but had no effect on senescence of ST-treated riceleaves. PEG or DH resulted in a marked increase in malondialdehyde (MDA)contentand decrease in superoxide dismutase (SOD) activity, but ST had no effect onMDAcontent and SOD activity. Treatment with NO donors caused a reduction of PEG-and DH-induced increase in MDA content and decrease in SOD activity. Theprotective effect of NO donors on promotion of senescence, increase in lipidperoxidation and decrease in SOD activity induced by PEG and DH was reversed by2-(4-carboxy-2-phenyl)-4,4,5,5- tetra-methylinmidazoline-1-oxyl-3-oxide, a NOspecific scavenger, suggesting that the protective effect of NO donors isattributed to NO released. The inhibition of PEG- and DH- enhanced senescenceofrice leaves by NO is most likely mediated through increasing SOD activity anddecrease in lipid peroxidation.     

杂交稻及其三系叶片衰老过程中SOD、CAT活性和MDA含量的变化

对杂交水稻及其三系主茎第11叶叶片自然衰老过程中超氧物歧化酶(SOD)、过氧化氢酶(CAT)和丙二醛(MDA)含量的变化进行了研究,结果表明:叶片衰老过程中,SOD和CAT活性下降,MDA的含量增加,可作为衰老特征的叶绿素和可溶性蛋白质含量明显下降;SOD的活性和MDA的含量变化相对应;CAT活性大幅度下降与SOD之间的不平衡,致使O_2~-代谢中间产物累积而引起膜的损伤。不育系的衰老进程比杂交水稻、恢复系和保持系慢,其SOD和CAT活性明显高于其它三者,可能是不育系不易早衰的原因之一。     

Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter.

Leaf senescence is an active process involving remobilization of nutrients from senescing leaves to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, supplemental cytokinin delays senescence. Plants that overexpress isopentenyl transferase (ipt), a cytokinin-producing gene, or knotted1 (kn1), a homeobox gene, have many phenotypes in common. Many of these phenotypes are characteristic of altered cytokinin physiology. The effect of kn1 on leaf senescence was tested by driving its expression using the promoter of the senescence-associated gene SAG12. SAG:kn1 tobacco plants showed a marked delay in leaf senescence but otherwise developed normally. The delay in senescence was revealed by an increase in chlorophyll content in SAG:kn1 leaves relative to leaves of the control plants and by a decrease in the number of dead leaves. Senescence was also delayed in detached leaves of SAG:kn1 plants. Delayed senescence was accompanied by increased leaf cytokinin content in older leaves expressing kn1. These experiments extend the current understanding of kn1 function and suggest that in addition to mediating meristem maintenance, kn1 is capable of regulating the onset of senescence in leaves.     

Ethylene and Cytokinin in the Control of Senescence in Detached Leaves of Arabidopsis thaliana eti-5Mutant and Wild-Type Plants

We studied the effects of cytokinin benzyladenine (BA) and ethylene on the senescence in the dark of detached leaves of Arabidopsis thaliana(L.) Heynh wild-type plants and theeti-5mutant, which was described in the literature as the ethylene-insensitive one. Leaf senescence was assessed from a decrease in the chlorophyll content. The content of endogenous cytokinins (zeatin and zeatin riboside) was estimated by the ELISA technique. We demonstrated that the content of endogenous cytokinins in the leaves of the three-week-old eti-5mutants exceeded that of the wild-type leaves by an order of magnitude; in the five-week-old mutants, by several times; and in the seven-week-old plants, the difference became insignificant. Due to the excess of endogenous cytokinins in the three–five-week-old mutant leaves, their senescence in the dark was retarded and exogenous cytokinin affected these leaves to a lesser extent. The seven-week-old mutant and the wild-type leaves, which contained practically similar amounts of endogenous cytokinins, did not differ in these indices. Thus, the level of endogenous cytokinins determined the rate of senescence and the leaf response to cytokinin treatment. Ethylene accelerated the senescence of detached wild-type leaves. Ethylene action increased with increasing its concentration from 0.1 to 100 l/l. BA (10^–6M) suppressed ethylene action. Similar data were obtained for the eti-5mutant leaves. We therefore suggest that the mutant leaves comprised the pathways of the ethylene signal reception and transduction, which provided for the acceleration of their senescence.     

Expression of Phospholipase D during Castor Bean Leaf Senescence

Membrane deterioration in plant senescence is commonly associated with progressive decreases in membrane phospholipid content. This study investigated the expression and regulation of phospholipase D (PLD; EC 3.1.4.4) during senescence in castor bean (Ricinus communis L. cv Hale) leaf discs. The rate of leaf senescence was accelerated by 50 [mu]M abscisic acid and was attenuated by 50 [mu]M cytokinin during incubation at 23[deg]C for up to 5 d. Leaf senescence was indicated by decreases in the content of total proteins, chlorophyll, and phospholipids. PLD activity in both membrane-associated and cytosolic fractions showed a gradual increase in the absence of phytohormones. Abscisic acid stimulated an increase in membrane-associated PLD and had little effect on the soluble form. On the other hand, cytokinin retarded the increase in membrane-associated PLD. Immunoblotting analysis using PLD-specific antibodies revealed that the changes in PLD activity were correlated with those of PLD protein. Analysis of PLD by nondenaturing PAGE showed the appearance of a PLD structural variant, PLD 3, in abscisic acid-treated leaf discs. Northern blotting analysis using a PLD cDNA probe revealed an increase in PLD mRNA in senescing leaf discs. These data indicate complex mechanisms for the regulation of PLD during senescence, which include increases in membrane-associated PLD, differential expression of PLD isoforms, and changes in amounts of PLD protein and mRNA. Such controlled expression points to a role for PLD in membrane deterioration and plant senescence.     

Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida

Changes in endogenous cytokinin content and cytokinin oxidase activity were characterized in leaf explants from two Petunia hybrida Vilm. genetic lines which differed in their shoot organogenic response to exogenous N⁶-benzyladenine (BA). Endogenous cytokinin content in leaf explants of the highly shoot organogenic line, St40, increased 1.7-fold during the shoot induction phase (days 6–10) and had an additional 2.6-fold cytokinin increase correlated with the shift from induction to the shoot development phase. The cytokinins isopentenyl adenine (iP) and isopentenyl adenosine (iPAR) increased, while the cytokinins zeatin, zeatin riboside and dihydrozeatin remained at consistently low levels. In contrast, isoprenoid cytokinins did not accumulate in petunia TLV1 leaf explants which were incapable of shoot induction during 12 days of culture with BA. Cytokinin oxidase activity continuously increased in leaf explants of both petunia genotypes in response to BA, with a larger increase in St40. These results suggest that the differences in organogenic response in the two petunia genotypes may be the result of differences in BA uptake and metabolism which subsequently affects the accumulation of isoprenoid cytokinins and the activity of cytokinin oxidase in the early stages of shoot development.     

Over-expression of Arabidopsis Bax inhibitor-1 delays methyl jasmonate-induced leaf senescence by suppressing the activation of MAP kinase 6

Methyl jasmonate (MeJA) is an important signalling molecule that has been reported to be able to promote plant senescence. The cell death suppressor Bax inhibitor-1 (BI1) has been found to suppress stress factor-mediated cell death in yeast and Arabidopsis. However, the effect and the genetic mechanism of Arabidopsis thaliana BI1 (AtBI1) on leaf senescence remain unclear. It was found here that the AtBI1 mutant, atbi1-2 (a gene knock-out), showed accelerated progression of MeJA-induced leaf senescence, while the AtBI1 complementation lines displayed similar symptoms as the WT during the senescence process. In addition, over-expression of the AtBI1 gene delayed the onset of MeJA-induced leaf senescence. Further analyses showed that during the process of MeJA-induced senescence, the activity of MPK6, a mitogen-activated protein kinase (MAPK), increased in WT plants, whereas it was significantly suppressed in AtBI1-overexpressing plants. Under the MeJA treatment, cytosolic calcium ([Ca(2+)](cyt)) functioned upstream of MPK6 activation and the elevation of [Ca(2+)](cyt) was reduced in AtBI1-overexpressing leaves. These results suggested a role of AtBI1 over-expression in delaying MeJA-induced leaf senescence by suppressing the [Ca(2+)](cyt)-dependent activation of MPK6, thus providing a new insight into the function and mechanism of AtBI1 in plant senescence.     

Cytokinin and anticytokinin activity of some 4-substituted 1H-pyrazoles and 8-aza analogues of adenine

Using specific bioassays i.e. radish cotyledon expansion, betacyanin synthesis in Amaranthus caudatus, and senescence retardation of isolated leaf explants, six 4-substituted 1-H pyrazoles and five 8-aza adenine analogues were tested for their cytokinin- and anticytokinin activity. Most of the pyrazole derivatives showed some cytokinin-like activity and enhanced the effect of 10^–5 M BA. 8-Aza substituted adenines were found to be cytokinin antagonists; in the bioassays used they were inactive when applied alone but blocked the action of 10^–5 M BA when applied simultaneously.     

Regreening of senescent Nicotiana leaves. I. Reappearance of NADPH-protochlorophyllide oxidoreductase and light-harvesting chlorophyll a/b-binding protein

Decapitation of Nicotiana rustica L. plants above a single senescent leaf induced regreening, which was promoted by cytokinin treatment. Regreening required low light. The decline in leaf protein content and increase in protease activity seen during senescence were reversed on regreening. Western blotting showed that light-harvesting chlorophyll a/b-binding protein declined considerably during senescence, but on regreening it increased back to the levels seen in green leaves. NADPH-protochlorophyllide oxidoreductase (POR) was found by Western blotting at high levels in etiolated cotyledons, but at low levels in green leaves and not at all in senescent leaves. However, POR reappeared in regreening leaves, and cytokinin accelerated its increase.     

厚朴叶片衰老过程中SOD对叶绿素、蛋白质及质膜透性效应的定量研究

本文应用弹性分析和边际分析数学方法对离体厚朴叶片衰老中的SOD活性对叶绿素、蛋白质及质膜透性效应进行了研究。结果表明:厚朴叶片衰老中,SOD活性对叶绿素或蛋白质或质膜透性的边际量和弹性系数的效应,是随着外源营养条件和温度变化而变化的。SOD活性与叶绿素及蛋白质的弹性系数呈显著正相关,SOD对它们的效应处于递增阶段,而SOD活性与质膜透性的弹性系数呈显著负相关,其效应处于负效应阶段。