Leaf senescence in a non-yellowing mutant of Festuca pratensis: Degradation of carotenoids

The loss of pigments was assessed in detached leaves of Festuca pratensis Huds. kept in permanent darkness. Two genotypes, a normal yellowing cultivar Rossa and a non-yellowing mutant Bf 993 were compared with each other. Analysis of individual pigments, chlorophylls. β-carotene, lutein, violaxanthin and neoxanthin was performed using HPLC. In the non-yellowing genotype the high retention of chlorophylls was associated with an equally high retention of total carotenoids. Although the two genotypes differ markedly with regard to the rate of pigment loss, the ratios of yellow to green pigments did not change significantly during dark-induced senescence. At the end of the senescence period β-carotene was retained to a higher degree than the xanthophylls, particularly in the yellowing genotype. In the mutant leaves the ratio of chlorophyll a to b remained nearly constant, whereas in leaves of the normal genotype a preferential retention of chlorophyll b was observed towards the end of the senescence period. It is concluded that the thylakoids of the non-yellowing genotype retain all the principal components of protein-pigment complexes, i.e. chlorophylls, carotenoids and apoproteins. Possible explanations for the stability of these complexes in the mutant are discussed.     

Observations on the Senescence of a Mutant Non-yellowing Genotype of Phaseolus vulgaris L.

A non-yellowing mutant of Phaseolus vulgaris L. was used toinvestigate factors involved in chlorophyll breakdown duringfoliar senescence. The mutant showed physiological changes similarto those of the normal yellowing type during senescence exceptthat leaf chlorophyll did not decline. Transmission electronmicroscope studies did not reveal appreciable differences inchloroplast ultrastructure between the two genotypes, suggestingthat chloroplast membrane integrity was not the factor preventingchlorophyll degradation in the mutant. However, the lack ofplastoglobuli in senescent mutant chloroplasts suggested thatthe lipid environment may be different from that of senescentnormal chloroplasts. Banding patterns of total soluble protein,resolved by sodium dodecyl sulphate-poly aery lamide gel electrophoresisshowed few, if any, differences between mature non-senescentnormal and mutant leaves; however, bands at 14 kD and 58 kDdiminished in senescent normal leaves, but remained in senescentmutant non-yellowing leaves. Key words: Non-yellowing mutant, Phaseolus vulgaris, senescence, chlorophyll degradation     

Analysis of the chlorophyll catabolism pathway in leaves of an introgression senescence mutant of Lolium temulentum

Pigments, proteins and enzyme activity related to chlorophyll catabolism were analysed in senescing leaves of wild-type (WT) Lolium temulentum and compared with those of an introgression line carrying a mutant gene from stay-green (SG) Festuca pratensis. During senescence of WT leaves chlorophylls a and b were continuously catabolised to colourless products and no other derivatives were observed, whereas in SG leaves there was an accumulation of dephytylated and oxidised catabolites including chlorophyllide a, phaeophorbide a and 13(2) OH-chlorophyllide a. Dephytylated products were absent from SG leaf tissue senescing under a light-dark cycle. Retention of pigments in SG was accompanied by significant stabilisation of light harvesting chlorophyll-proteins compared with WT, but soluble proteins such as Rubisco were degraded during senescence at a similar rate in the two genotypes. The activity of phaeophorbide a oxygenase measured in SG tissue at 3d was less than 12% of that in WT tissue at the same time-point during senescence and of the same order as that in young pre-senescent WT leaves, indicating that the metabolic lesion in SG concerns a deficiency at the ring-opening step of the catabolic pathway. In senescent L. temulentum tissue two terminal chlorophyll catabolites were identified with chromatographic characteristics that suggest they may represent hitherto undescribed catabolite structures. These data are discussed in relation to current understanding of the genetic and metabolic control of chlorophyll catabolism in leaf senescence.     

What stay-green mutants tell us about nitrogen remobilization in leaf senescence

Leaf senescence has an important role in the plant's nitrogen economy. Chlorophyll catabolism is a visible symptom of protein mobilization. Genetic and environmental factors that interfere with yellowing tend to modify protein degradation as well. The chlorophyll-protein relationship is much closer for membrane proteins than it is for soluble or total leaf proteins. In stay-greens, genotypes with a specific defect in the chlorophyll catabolism pathway, soluble protein degradation during senescence may be close to normal, but light-harvesting and reaction centre thylakoid membrane proteins are much more stable. Genes for the chlorophyll catabolism pathway and its control are important in the regulation of protein mobilization. Genes for three steps in the pathway are reported to have been isolated. The gene responsible for the stay-green phenotype in grasses and legumes has not yet been cloned but a fair amount is known about it. Pigment metabolism in senescing leaves of the Festuca-Lolium stay-green mutant is clearly disturbed and is consistent with a blockage at the ring-opening (PaO) step in chlorophyll breakdown. PaO is de novo synthesized in senescence and thought to be the key enzyme in the chlorophyll a catabolic pathway. The stay-green mutation is likely to be located in the PaO gene, or a specific regulator of it. These genes may well be in the various senescence-enhanced cDNA collections that have been generated, but functional handles on them are currently lacking. When the stay-green locus from Festuca pratensis was introgressed into Lolium temulentum, a gene encoding F. pratensis UDPG-pyrophosphorylase was shown to have been transferred on the same chromosome segment. A strategy is described for cloning the stay-green gene, based on subtractive PCR-based analyses of intergeneric introgressions and map-based cloning.     

晚播小麦叶片衰老代谢和粒重变化的比较研究

对７个小麦品种在晚播条件下的叶片衰老生理特性和粒重变化进行了比较研究。根据小麦叶片的衰老特征相差差异,将７个小麦品种区分为３个类型;后健型、早衰型和中间型。在小麦旗叶的衰老过程中,后健型小麦品种旗叶叶绿素和类胡萝卜素含量显著高于早衰型,脂质过氧化产物ＭＤＡ含量显著低于早衰型,小麦粒重降幅依次为早衰型〉中间型〉后健型,并讨论了活性氧代谢在小麦叶片衰老过程中可能作用。     

Chloroplast pigments,proteins, lipid peroxidation and activities of antioxidative enzymes during maturation and senescence of leaves and reproductive organs of Cajanus cajan L.

A comparative investigation was undertaken with pigeon pea leaves and attached flower buds/flowers/pods during their developmental stages including senescence in a natural system in experimental plots. Alterations in chloroplast pigments, total soluble proteins, lipid peroxidation, malondialdehyde (MDA) content and activities of guaiacol peroxidase (POD, EC 1.11.1.7) and superoxide dismutase (SOD, EC 1.15.1.1) were studied at 5-day interval from initial to 40-day stage. Chloroplast pigments and proteins of leaves increased upto 15 and 20-day stages respectively followed by a steady decline. Reproductive parts, however, exhibited rise in chloroplast pigments upto 25-day and protein till last stage as developing pods gain the amount from the senescing leaves which are nearest to them. Senescing leaves show very high POD activity than the developing and senescing pods and POD appears to be associated with chlorophyll degradation. Considerably higher activity and amount of LOX and MDA respectively have been noticed in senescing leaves than in flowers and pods. Increase in SOD activity during early stage of leaf growth and maturation indicates protective role that declined at senescent stages. Pods are unique in having very high SOD activity, only last stage of senescence does show a decline.     

Leaf Senescence and Abscisic Acid in Leaves of Field-grown Soybean

Leaf senescence in field-grown soybean (Merrill) as defined by the period after full expansion, was studied by measuring abscisic acid (ABA), total soluble protein, and chlorophyll in leaves through the later part of the growing season. ABA concentrations increased significantly at the end of the season when leaves had started to turn yellow, well after total soluble protein and chlorophyll had started to decline. The results indicate that events occurring before leaf yellowing are more significant in evaluating leaf senescence since the yellowing condition and rise in ABA are effects of changes in physiological activity beginning when leaves are still green.     

春玉米叶片衰老中激素变化,Ca^2＋跨膜运输和膜脂过氧化三者之间的关系

通过离体玉米（ZeamaysL．）叶片培养和叶肉质膜微囊45Ca2 吸收等实验,探索春玉米叶片衰老过程中激素变化、Ca2 跨膜运输及膜脂过氧化三者之间的联系。结果认为,玉米叶片衰老的可能过程首先是内源激素含量变化,继而影响到Ca2 跨膜运输,进而导致膜脂过氧化,由此引起叶绿素和蛋白质降解。     

Nondestructive analysis of senescence in mesophyll cells by spectral resolution of protein synthesis-dependent pigment metabolism

* Over 6 d of dark-induced senescence, leaf segments of wild-type Lolium temulentum lost > 96% chlorophyll a + b; leaves from plants containing a staygreen mutation introgressed from Festuca pratensis, which has a lesion in the senescence-associated fragmentation of pigment-proteolipid complexes, retained over 43% of total chlorophyll over the same period. * Mutant segments preferentially retained thylakoid membrane proteins (exemplified by LHCP II) but lost other cellular proteins at the same rate as wild-type tissue. The protein synthesis inhibitor D-MDMP inhibited chlorophyll degradation and partially prevented protein loss in both genotypes, but tissues treated with the ineffective L-stereoisomer were indistinguishable from water controls. * Principal-components analysis of leaf reflectance spectra distinguished between genotypes, time points and D-MDMP treatments, showing the disruption of pigment metabolism during senescence brought about by the staygreen mutation, by inhibition of protein synthesis and by combinations of the two factors. * The build-up of oxidized, dephytylated and phaeo-derivatives of chl a during senescence of staygreen tissue was prevented by D-MDMP and associated with characteristic difference spectra when senescent mutant tissue was compared with wild-type or inhibitor-treated samples. The suitability of senescence as a subject for systems biology approaches is discussed.     

Increases in Cytosolic Ca2+ in Parsley Mesophyll Cells Correlate with Leaf Senescence

The ability to maintain the cytoplasmic Ca2+ concentration ([Ca2+]cyt) at homeostatic levels has been examined during leaf senescence in detached parsley (Petroselinum crispum) leaves. Fluorescence ratiometric imaging of mesophyll cells isolated from parsley leaves at various senescence stages and loaded with the Ca2+ indicator fura-2 has revealed a distinct elevation of [Ca2+]cyt, which was positively correlated with the progress of leaf senescence. This initial increase of [Ca2+]cyt, which was first observed in cells isolated from 3-d-senescent leaves, occurred 1 d before or in parallel with changes in two established senescence parameters, chlorophyll loss and lipid peroxidation. However, the [Ca2+]cyt elevation followed by 2 d the initial increase in the senescence-associated proteolysis. Whereas the [Ca2+]cyt of nonsenescent cells remained at the basal level, the elevated [Ca2+]cyt of the senescent cells was a long-lasting effect. Experimental retardation of senescence processes, achieved by pretreatment of detached leaves with the cytokinin benzyladenine, resulted in maintenance of homeostatic levels of [Ca2+]cyt in cells isolated from 3-d-senescent leaves. These observations demonstrate for the first time to our knowledge a correlation between elevated [Ca2+]cyt and the process of senescence in parsley leaves. Such senescence-associated elevation of [Ca2+]cyt, which presumably results from a loss of the cell's capability to extrude Ca2+, may serve as a signal inducing subsequent deteriorative processes.     

Relationships Among Hormone Changes,Transmembrane Ca2+ Flux and Lipid Peroxidation During Leaf Senescence in Spring Maize

45Ca2+ absorption by plasma membrane vesicles isolated from maize (Zea mays L. ) leaves cultured in vitro was assessed to investigate the relationships among hormone changes, transmembrane Ca2+ flux and lipid peroxidation during leaf senescence. The results suggested that the possible process of leaf senescence in spring maize followed a sequence of endogenous hormonal change then transmembrane Caa + flux which further led to lipid peroxidation, with consequent degradation of chlorophyll and protein.     

EFFECT OF PETIOLE PHLOEM DISRUPTION ON STARCH AND MINERAL DISTRIBUTION IN SENESCING SOYBEAN LEAVES

Normally, starch (sugars) and minerals are redistributed from the leaves to the pods during monocarpic senescence in maturing soybean plants. Petiole phloem destruction (steam girdling), which blocked this redistribution by interrupting export through the petiole, altered the foliar senescence pattern producing a distinctive interveinal yellowing with green areas along the veins on pod-bearing plants. This suggests that blockage of the petiole phloem may cause nutrients to accumulate in the green zones along the leaf veins instead of being redistributed to the pods. In the leaves of untreated plants, starch showed the same distribution pattern as chlorophyll; however, starch was preserved in yellow areas as well as the green zones of the steam-girdled leaves. Mineral analyses of the veinal and interveinal zones of treated leaves and controls showed that the veinal green zones and interveinal yellowing in treated plants were not respectively enriched and depleted in minerals corresponding to a redistribution of minerals within the leaves. Depodding also blocked leaf yellowing, net mineral redistribution and starch breakdown. Thus, the pods are able to induce chlorophyll breakdown without net mineral redistribution or starch loss in leaves with petiole phloem destruction. This shows that chlorophyll breakdown is not obligatorily coupled with mineral redistribution or starch breakdown.     

The Effect of Light and Hormones on Leaf Senescence

With wheat leaves as material, the changes of superoxide dismutase (SOD), lipid peroxi-dation and membrane permeability during leaf senescence in light or dark, and treated withphytohormones (KT or ABA) have been studied. The changes of chlorophyll content, lipidperoxidation and fine structure of spinach chloroplasts senescing in light or dark have alsobeen studied. When leaves senesce in light, the activity of SOD increased at first then decreased. The increase of SOD activity was able to result from the synthesis of new protein. Lightwas found to delay the leaf senescence obviously but also accelerate leaf senescence by causinglipid peroxidation when prolonged the illumination time. The delay or acceleration of leafsenescence by exogenous hormones were observed, it may be due to the control of lipid peroxi-dation by adjusting the activity of SOD. O2-participated the chlorophyll decomposition andlipid peroxidation during chloroplasts senesce in light. A favourable role of light in mainta-lng the fine structure of isolated chloroplasts was clear.     

水稻叶片的衰老与超氧物歧化酶活性及脂质过氧化作用的关系

研究了从抽穗开花到籽粒成熟过程中,水稻植株顶部三片叶子的超氧物歧化酶(SOD),脂质过氧化产物丙二醛(MDA)含量及二磷酸核酮糖羧化酶活性的变化。实验结果表明:叶片的衰老伴随着 SOD 活性、RuBP 羧化酶活性及叶绿素含量的降低、丙二醛含量显著增高。分离了三个 SOD 的同工酶,证明为 Cu—Zn SOD。观察了 SOD 同工酶在叶片老化及酶液存放不同时间中的变化。讨论了叶片衰老过程中氧自由基对酶及质膜的损伤影响。     

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。     

Chlorophyll a fluorescence, enzyme and antioxidant analyses provide evidence for the operation of alternative electron sinks during leaf senescence in a stay-green mutant of Festuca pratensis

Mutation of the sid gene in Festuca pratensis prevents chlorophyll degradation. The senescing leaves retain their chlorophyll complement and stay green. Nevertheless, CO2 assimilation and ribulose-bisphosphate carboxylase/oxygenase content decline in both mutant and wild-type plants. Photosynthesis and chlorophyll a fluorescence measurements were performed in air and at low oxygen to prevent photorespiration. The maximum extractable activity of ribulose 1,5 bisphosphate carboxylase was higher in the senescent mutant leaves than in those of the wild-type control hut Mas much lower than that observed in the mature leaves of either genotype. The activation state of this enzyme was similar in mutant and wild-type lines at equivalent stages of development. Analysis of chlorophyll a fluorescence quenching with varying irradianco showed similar characteristics for mature leaves of the two genotypes. Genotypic variations in photosystem II (I'SII) efficiency were observed only in the senescent leaves. Photochemical quenching and the quantum efficiency of PSII were greater in the senescent mutant leaves than in (he wild type at a given irradiance. The calculated electron flux through PSII was substantially higher in the mutant with a greater proportion of electrons directed to photorespiration. Maximum catalytic activities of ascorbate peroxidase decreased in senescent compared to mature leaves of both genotypes, while glutathione reductase and monodehydroascorbate reductase were unchanged in both cases. Superoxide dismutase activity was approximately doubled and dehydroascorbate reductase activity was three times higher in senescent leaves compared with the mature leaves of both genotypes. In no case was there a difference in enzyme activities between mutant and wild type at equivalent growth stages. The pool of reduced ascorbate was similar in the mature leaves of the two genotypes, whereas it was significantly higher in the senescent leaves of the mutant compared with the wild type. Conversely, the hydrogen peroxide content was significantly higher in the mature leaves of the wild type than in those of the mutant, but in senescent leaves similar values were obtained. In leaves subjected to chilling stress the reduced ascorbate pool was higher in both mature and senescent leaves of the mutant than in their wild-type counterparts. Similarly, the hydrogen peroxide pool was significantly lower in both mature and senescent leaves of the mutant than in the wild type. We conclude that, in spite of deceased CO₂ assimilation, the mutant is capable of high rates of electron Slow. The high ascorbate/hydrogen peroxide ratio observed in the mutant, particularly at low temperatures, suggests that the senescent leaves are not subject to enhanced oxidative stress.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.Abbreviations kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b-binding protein - M_r relative molecular mass - PSII photosystem II - SDS sodium dodecyl sulphate     

Superoxide Dismutase Activity and Lipid Peroxidation in Relation to Senescence of Rica Leaves

Superoxide dismutase (SOD), lipid peroxidation, ribulose 1, 5-bisphosphate earboxylasc and chlorophyll content in the leaves of rice were investigated during bloomJug to ripening stages. The results indicated that leaf senescence was associated with the decreascs of SOD and RuBP earboxylase activities and chlorophyll content. A marked increase of malondialdehyde(MDA)contcnt, a produet of lipid pcroxidation was observed during leaf senescence, However, the relative high activities of SOD and RuBP earboxylase and chlorophyll content, lower MDA content in hybrid rice senescenee leaves, compared with their three lines, suggest the presence of physiological hybrid vigor. The analysis of polyacrylamide gel eleetrophoresis showed that three SOD isozymes in leaf extract, which were Cu-Zn SOD. Changes of SOD isozymes were observed during leaf agling and at different time of storage of its extract. The destroying effect of oxygen free radical on enzymes and membrane in the course of leaf senescence was discussed.