Effects of Light Illumination on the Population of Translatable mRNA in Radish Cotyledons during Dark-Induced Senescence

The population of translatable mRNA in radish cotyledons hasbeen shown to change strikingly during dark-induced senescenceby analyzing cell-free translation products from polyadenylatedRNA using two-dimensional electrophoresis (Kawakami and Watanabe1988, Plant Cell Physiol. 29: 33–42). In the present study,the translatable mRNAs in the cotyledons were studied afterre-illumination of seedlings which had been induced to senesceby dark treatment. All of the mRNA species which showed relativeincrease or decrease during a 24-h dark treatment returned totheir initial levels after a subsequent 24-h illumination withwhite light. When seedlings were kept in the dark for 48 h,21 mRNA species increased, but the increase in some of themwas no longer reversible; 9 out of the 21 mRNA species remainedat the same level even after 48 h of re-illumination. All ofthe 18 mRNA species which decreased during 48 h in the darkreturned to their initial levels upon re-illumination. The mRNAspecies that showed irreversible increase were also found toaccumulate in cotyledons senesced after a lengthy growth periodunder light. There were also some mRNA species whose contentwas not affected by dark treatment, but increased transientlyafter re-illumination of the seedlings. These observations arediscussed in relation to the progress of senescence in the cotyledons. (Received September 24, 1987; Accepted December 19, 1987)     

萝卜离体子叶衰老与膜脂过氧化的关系

萝卜离体子叶在光下或暗中衰老及激素调节衰老过程中,作为叶片衰老指标的叶绿素和蛋白质含量的降低,发生在MDA含量增高之前,更早于SOD活性的下降。表明由SOD活性降低所导致的膜脂过氧化的增强,并非衰老的原初反应,而是叶片衰老到一定程度的生理变化。因此,至少在萝卜离体子叶上,不能将其衰老的启动归因于受SOD控制的膜脂过氧化作用导致的膜累积性质变。     

Change in Gene Expression in Radish Cotyledons during Dark-Induced Senescence

Cotyledons detached from light-grown radish (Raphanus sativusL. cv. Comet) seedlings were used as a model system to studythe changes in nuclear gene expression during dark-induced senescenceof green leaves. Polyadenylated RNA was prepared from the cotyledonsat different times and then translated in a wheat germ system.Approximately 1,000 different polypeptides of the translationproducts were separated from each other by two-dimensional gelelectrophoresis. As judged from the density of autoradiographicspots of the translation products, the induction of senescenceby dark treatment involved an increase in 26 species, a decreasein 11 species, and a temporary increase and subsequent decreasein 8 species of translatable mRNA. A similar pattern of changein protein synthesis was also observed in the dark-treated cotyledonswhen the cotyledons were pulse-labeled with ³⁵S-methionine andthe soluble proteins separated by two-dimensional gel electrophoresis,though the polypeptide pattern on the gel did not coincide exactlywith those of the cell-free translation products. These findingsstrongly suggest that the process of leaf senescence is notsimply a passive and gradual death of the tissue, but involvesa drastic and sequential response of the cells to environmentalstimuli with respect to the gene expression of the cells. (Received July 21, 1987; Accepted September 30, 1987)     

Delay of Senescence of Detached Cucumber Cotyledons by Triadimefon

Changes in contents of reactive oxygen species (O₂ ⁻ and H₂O₂) and non-enzymatic antioxidants, activities of antioxidant enzymes and lipid peroxidation were investigated during senescence of detached cucumber cotyledons dipped in water (control) and 20 mg dm⁻³ triadimefon (TDM). O₂ ⁻ and H₂O₂ accumulation and lipid peroxidation were observed during senescence of cucumber cotyledons, which coincided with a drop in the contents of carotenoids (Car) and ascorbic acid (AsA), and the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), and an increase in the activity of peroxidase (POD). However, TDM could significantly inhibit the accumulation of O₂ ⁻ and H₂O₂, and lipid peroxidation by preventing the decrease of CAT, APX, Car and AsA and the increase of POD, while TDM had little effect on SOD activity during the senescence. Therefore we can draw a conclusion that TDM protects the membrane system and retards the senescence of detached cucumber cotyledons. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Effect of Light on the Senescence of Detached Cucumber Cotyledons

Measurements of cell constituents of cotyledons of cucumberexcised and kept in the light show differences from those excisedand kept in the dark. These differences are partly resolvedby adding glucose to the water-supply of the cotyledons in thedark suggesting that a lack of photosynthetic products is thecontrolling factor in their senescence. Under other circumstancesdifferent factors are operative suggesting that the first stepin senescence is different under different conditions.     

Effects of Sucrose on Rooting and Senescence in Detached Raphanus sativus L. Cotyledons

Application of 0.5–4.0 per cent sucrose to excised radishcotyledons incubated in the light suppressed the formation ofroots, enhanced the rate of yellowing, and altered the locationof chlorophyll loss from the margin of the lamina to the petiolarregion in most of the cotyledons. These effects were not producedwhen osmotica such as mannitol or polyethylene glycol were used. A few cotyledons were able to root in the presence of sucroseand these showed enhanced yellowing at the margin. The numberof such cotyledons increased when they were incubated in waterfor the period during which primordium initiation begins, beforetransfer to sucrose. However, the pattern of yellowing in thepresence of sucrose was not affected by the repeated removalof the region of primordium development, suggesting that thepattern of senescence is not controlled by the formation ofroot primordia. The results are discussed in relation to the effects of lightand possible accumulation of carbohydrate on senescence androot formation in excised tissues.     

Changes in Properties of Succinate Dehydrogenase during Senescence of Pea Cotyledons

Comparisons were made between succinate dehydrogenases (EC 1.3.99.1 [EC] )from 1-day-old and 5-day-old pea cotyledons. The enzyme wasloosely bound to the mitochondrial inner membrane in 5-day-oldcotyledons, but tightly in 1-day-old cotyledons. In addition,the enzyme partially purified from 5-day-old cotyledons wasmuch more labile than that from 1-day-old cotyledons. Succinaterapidly inactivated partially purified succinate dehydrogenasefrom 1-day-old cotyledons, but not from 5-day-old cotyledons.Dithiothreitol caused a change in the charge of the enzyme proteinfrom either 1- or 5-day-old cotyledons, only when succinatewas present. The enzyme from 5-day-old cotyledons differed fromthe succinate-induced labile form of the enzyme from 1-day-oldcotyledons in electrophoretic properties on a polyacrylamidegel. There was also a difference in the pattern of polyacrylamidegel electrophoresis between succinate dehydrogenases partiallypurified from 1- and 5-day-old cotyleodns. The partially purifiedenzyme from either 1- or 5-day-old cotyledons in the presenceof succinate had a molecular weight of 92,000. The molecularweight of the large subunit was suggested to be 65,000. Thepartially purified enzyme prepared from 1-day-old cotyledonsin the absence of succinate was in a form with a molecular weightof 113,000. (Received August 29, 1980; Accepted December 3, 1980)     

Hormonal Aspects of Senescence in Detached Tobacco Leaves

The objective of the present work was to describe the simultaneous changes in endogenous levels of cytokinins, abscisic acid, indoleacetic acid and ethylene in detached, senescing tobacco (Nicotiana rustica L.) leaves. These measurements were related to changes in chlorophyll contents, ¹⁴CO₂ fixation and proline contents — three parameters which have been considered to reflect senescence. Effects of exogenous hormonal treatments on these parameters, as well as on endogenous hormonal levels, provided further evidence for the interrelationships between hormones and for their roles in senescence. Starting with actively growing attached leaves and ending with well-advanced senescence in detached leaves, our data indicate a chronological sequence of three hormonal states: (a) cytokinins — high activity, abscisic acid, auxin and ethylene — low contents (actively growing, attached leaves); (b) cytokinins — low activity, abscisic acid — high, auxin and ethylene — low contents (apparent induction of senescence in detached leaves); and (c) cytokinins and abscisic acid — low, auxin and ethylene — high contents (senescence proper in detached leaves).     

Differential Induction of Endoproteinases during Senescence of Attached and Detached Barley Leaves

Endoproteinase activities and species were compared during dark-induced senescence of attached and detached primary barley leaves by isoelectric focusing and polyacrylamide gel electrophoresis of cell-free extracts. Neither of the two major endoproteinases (EP1 and EP2) changed in amounts during senescence of attached leaves, nor did new endoproteinases appear. In contrast, during senescence of detached leaves, both EP1 and EP2 activities increased and four new species of endoproteinases appeared. Proteolytic activity was evenly distributed throughout attached leaves, but activity in the detached leaf increased sharply from the tip to the base with the four new higher molecular weight species of proteinases present only in the bottom half of the leaf nearest the cut end. Thus a wound response may be superimposed on the processes of senescence in detached leaves. Cycloheximide and kinetin both inhibited the increase of EP1, EP2, and the induction of the four new endoproteinases; chloramphenicol had no effect. Indications are that both the increases in activity and the induction of new species of proteinases were the result of activity of cytoplasmic ribosomes.

Hydrolysis of total protein and ribulose-1,5-bisphosphate carboxylase protein in vivo was somewhat faster in detached than attached leaves. The difference, however, was much less than would be expected from the great increase in proteolytic activity in detached leaves.

     

Developmental Changes in Levels of Translatable mRNAs for Enolase Isozymes in Chicken Brain

Using chicken brain mRNAs, alpha and gamma enolase precursors were synthesized in the rabbit reticulocyte cell-free translation system. The product proteins showed molecular weights almost identical to those of the mature subunits. The levels of translatable mRNAs for alpha and gamma subunits were determined by the cell-free translation system and immunoprecipitation with specific antisera, during development of chicken brain. The level of alpha mRNA was high at any developmental stage of the brain. On the other hand, the gamma mRNA level was very low at the early embryonic stage, and increased rapidly during development of the brain. These changes were closely correlated with those of the corresponding enzyme activities, indicating that the levels of enolase activities in developing brain were controlled primarily by the level of the translatable alpha and gamma mRNAs.     

Development of Enzymes of the Glyoxylate Cycle during Senescence of Pumpkin Cotyledons

The presence and activities of isocitrate lyase (EC 4.1.3.1 [EC] )and malate synthase (EC 4.1.3.2 [EC] ) were studied during senescenceof pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). Afterincubation of detached cotyledons in permanent darkness, theactivities appeared and increased up to the eighth day and thendeclined, while the activities of catalase (EC 1.11.1.6 [EC] ), glycolateox-idase (EC 1.1.3.1 [EC] ), and hydroxypyruvate reductase (EC 1.1.1.81 [EC] )decreased dramatically. After fractionation of cell organellesby sucrose density gradient, we detected isocitrate lyase andmalate synthase activities in peroxisomal fractions. The activityof the two key enzymes of the glyoxylate cycle also increasedduring senescence in vivo and we confirmed the presence of thetwo enzymes in the peroxisomal fractions after sucrose gradientcentrifugation. At every point examined, the level of malatesynthase was demonstrated by immunoblotting. It is concludedthat the development of isocitrate lyase and malate synthaseactivities represents the transition from leaf peroxisomes toglyoxysomes and that such a phenomenon is associated with senescence. (Received January 25, 1991; Accepted March 22, 1991)     

Membrane Permeability--Regulation by Exogenous Sugars during Senescence of Oat Leaf in Light and Darkness

The effect of sugars (sucrose, glucose and fructose) on normalphysiological changes during senescence of foliar segments ofAvena sativa cv. Suregrain was studied. In general applicationof sugars raised tissue permeability both in the light and indarkness. This change was associated with increases in endogenoussugars, hydroperoxide content and lipoxygenase activity. Inthe light it was also associated with low catalase activity.Sugars did not influence superoxide dismutase activity. In thelight, sugars accelerated senescence, measured as decreasesin chlorophyll and increases in soluble amino acids. In darknesssugars delayed senescence. The effect of sugars in the lightseemed to result from an increase in photo-oxidations associatedwith the increase in permeability. The delaying effect on senescence,found in darkness, seemed to result from an increase in respiratoryactivity plus the lack of (or combined with the lack of) photo-oxidations. (Received March 18, 1985; Accepted June 3, 1986)     

Behavior of Lipoxygenase during Establishment, Senescence, and Rejuvenation of Soybean Cotyledons

Lipoxygenase protein and activity were examined during establishment, senescence, and rejuvenation of soybean cotyledons. Lipoxygenase protein, as determined on `Western' immunoblots, and lipoxygenase-1 and -2/3 activities decreased during mobilization of seed reserves 3 to 9 days following planting. Lipoxygenase-1 activity decreased more rapidly than lipoxygenase-2/3 and was not detectable by 11 days after planting. Lipoxygenase protein increased after day 11 while lipoxygenase-2/3 activity continued to decline. During the later stages of cotyledon senescence, both lipoxygenase protein and lipoxygenase-2/3 activity decreased. Upon rejuvenation, lipoxygenase-2/3 activity, but not that of lipoxygenase-1, increased. These results demonstrate that elevated lipoxygenase activity does not represent a universal characteristic of senescent plant tissue.     

Changes in Free Fatty-acid Content and Respiratory Activity during the Senescence of Cotyledons of Cucumber

During senescence the amount of free fatty acid in the chloroplastdecreases while the level in the tissue as a whole increases.Although this rise is sufficient to bring about an inhibitionof mitochondrial activity during the final stages of senescenceit occurs too late to explain the initial respiratory declinein cucumber cotyledons.     

Calcium- and Calmodulin-Promoted Phosphorylation of Membrane Proteins during Senescence in Apples

Sodium dodecylsulfate-polyacrylamide gel electrophoresis ofmicrosomal membrane proteins from post-climacteric apples atan early and an advanced stage of senescence showed only slightqualitative changes in the protein pattern. Though there wasa 30% reduction in the total microsomal protein content in applesat an advanced stage of senescence, a polypeptide with 18,000molecular weight increased in quantity during senescence. Invitro phosphorylation of several proteins was promoted by calciumin membranes from apples at an early stage of senescence. Phosphorylationof proteins with molecular weights of 95,000, 91,000, 53,000and 50,000 was promoted by calcium and calmodulin. Phosphorylationof these proteins increased with increasing calcium concentration.Proteins with molecular weights of 53,000 and 50,000 showedmarked promotion of phosphorylation over the calcium-promotedlevel when the amount of calmodulin in the assay mixture wasincreased. Calcium- and calmodulin-promoted phosphorylationof membrane proteins showed considerable decrease when the appleswere at an advanced stage of senescence. Moreover, increasingthe concentrations of calcium and calmodulin in the assay mixturedid not have any promoting effect on the phosphorylation ofthese proteins. Phosphoprotein phosphatase activity as measuredby the loss of label from phosphorylated proteins followingchase with cold ATP, did not differ to a great extent in membranepreparations from normal and senesced apples. Hydrolysis ofATP by senesced apple membrane preparation, however, was foundto be relatively higher. The significance of these observationsin relation to senescence is discussed. ¹ Scientific Paper No. 7084, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321.² Supported in part by grants from the Washington State TreeFruit Research Commission, and National Science Foundation GrantPCM-8208408.     

Induction of 33-kD and 60-kD Peroxidases during Ethylene-Induced Senescence of Cucumber Cotyledons

Ethylene enhanced the senescence of cucumber (Cucumis sativus L. cv `Poinsett 76') cotyledons. The effect of 10 microliters per liter ethylene was inhibited by 1 millimolar silver thiosulfate, an inhibitor of ethylene action. An increase in proteins with molecular weights of 33 to 30 kilodaltons and lower molecular weights (25, 23, 20, 16, 12, and 10 kilodaltons) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after ethylene enhanced senescence. The measurement of DNase and RNase activity in gels indicated that these new proteins were not nucleases. Two proteins from ethylene-treated cotyledons were purified on the basis of their association with a red chromaphore and subsequently were identified as peroxidases. The molecular weights and isoelectric points (pI) of two of these peroxidases were 33 kilodaltons (cationic, pI = 8.9) and 60 kilodaltons (anionic, pI = 4.0). The observation that [³⁵S]Na₂SO₄ was incorporated into these proteins during ethylene-enhanced senescence suggests that these peroxidases represent newly synthesized proteins. Antibodies to the 33-kilodalton peroxidase precipitated two in vitro translation products from RNA isolated from ethylene-treated but not from control cucumber seedlings. This indicates that the increase in 33-kilodalton peroxidase activity represents de novo protein synthesis. Both forms of peroxidase degraded chlorophyll in vitro, which is consistent with the hypothesis that peroxidases have catabolic or scavenging functions in senescent tissues.     

萌发绿豆子叶衰老期间蛋白质代谢的变化

萌发绿豆子叶自然衰老过程中可溶性蛋白质含量一直下降;从衰老开始到衰老前期,总游离氨基酸含量明显上升;但游离氨基酸各组分在子叶衰老期间的变化趋势并不相同。~3H-亮氨酸掺入蛋白质试验和多聚核糖体的相对量及其与总核糖体的比值(P/T)测定都证明在子叶衰老前期有蛋白质的新合成。子叶衰老期间。氨肽酶活性明显降低;而以酪蛋白为底物的蛋白水解酶活性却急剧上升,承担着催化蛋白质降解的主要功能。     

萝卜带柄子叶高频再生体系的建立

以11份萝卜（Raphanus sativus）基因型为材料进行子叶离体培养研究,筛选出具有较高再生率的基因型进行实验,考察基因型、外植体类型、激素配比和苗龄等因素对萝卜再生的影响。结果表明：萝卜离体再生的最佳外植体为全子叶-叶柄,最适苗龄为4天,最适培养基为MS＋6mg·L-16-BA＋0.05mg·L-1NAA,再生率高达86.95%,再生系数为1.80。该研究为进行萝卜遗传转化实验奠定了良好基础。     

Root Hypoxia-lnduced Changes in the Pattern of Translatable mRNAs In Poplar Leaves

Shoot responses to root stress may be modulated by hormonestransported from root tissues. In this study, the pattern ofin vitro translatable mRNAs from leaves on plants with oxygen-deprived(hypoxic) roots was used as a molecular phenotype to test forthe action and specificity of putative root-to-shoot signalmolecules. Root hypoxia caused a decrease in the abundance ofa group of leaf mRNAs which translated to give low molecularweight (LMW) proteins (12 to 28 kDa). Root excision induceda similar reduction in a subset of the same LMW leaf mRNAs.This suggests that the effects of root hypoxia on leaves arephysiologically related to the effects of root excision. Feedingabscisic acid to leaves did not mimic the effects of root aerationor hypoxia. Applications of zeatin riboside (ZR) to leaves ofplants with hypoxic roots duplicated the effects of root aerationon the LMW translation products. The specificity of the leafresponse to ZR was tested by feeding KC1 and sucrose to leavesof root excised plants. Applications of either KC1 or sucrosealso increased the abundance of the LMW translation products.Although the results reported in this paper are consistent withthe concept that shoot responses to root stress may be modulatedby substances moving in the xylem, a thorough understandingof how hormones control cellular responses needs to be resolvedbefore the mechanism of root-shoot integration can be understood. Key words: ABA, cytokinins, hypoxia, poplar