Changes in French bean cotyledon composition associated with modulated life-span

The onset of Phaseolus vulgaris L. cotyledon senescence and its characteristics were modulated by irradiance (higher or lower than standard) and by epicotyl decapitation. The cotyledon life-span of 16 d was not influenced by irradiance while decapitation prolonged the life-span to 28 d. The fresh mass of cotyledons, an indicator of organ viability, decreased in a similar manner in all non-decapitated plants, though it was relatively slower in plants grown under a low irradiance (LI). Three days after decapitation the fresh mass of cotyledons increased by one third, a slight decrease was observed on the 21st d, and it lasted until the end of the life span. Deducing from the fall of chlorophyll (Chl) concentration expressed per unit protein, senescence started after the 10th day in non-decapitated plants. Decapitation postponed the onset of senescence until the 21st day. Expression of Chl amount per unit dry mass did not detect any changes in LI plants, hence this parameter can not be used for the assessment of senescence. The measurements of Chl a and b concentrations indicated that the light-harvesting complexes (LHCs) proliferated during ageing and were rapidly destroyed at the onset of senescence. Changes of the concentrations of carotenoids supported the hypothesis of free radicals involvement in senescence. The bean cotyledons responded to free radical production induced under higher irradiance by increased β-carotene synthesis. Oxidative damage to galactolipids during senescence was documented by fluorescence measurements. The changes in cotyledon composition were correlated to morphologic changes observed by electron microscopy.     

The capacity of antioxidant protection during modulated ageing of bean (Phaseolus vulgaris L.) cotyledons. 1. The antioxidant enzyme activities

Reactive oxygen species are known to increase in plant senescence. We investigated the participation of antioxidative enzymes in initiation of cotyledon senescence. Senescence of bean (Phaseolus vulgaris L.) cotyledons was modulated by UV C irradiation and by the decapitation of plant apices. Senescence was accompanied by a decrease of protein content and by a decrease of photochemical efficiency. A drop in activity of antioxidative enzymes preceded the onset of senescence in control plants. In cotyledons with prolonged life span, the decrease of antioxidant activities and the markers of senescence onset appeared at a similar age as in controls. Thus we presumed that the period from senescence initiation to cotyledon abscission was extended. On the other hand, in UV C irradiated plants we did not observe actual senescence initiation, and antioxidant enzymes although elevated, did not effectively play their role. The decrease of antioxidant enzymes activity and the markers of senescence appeared at a similar age both in control and in decapitated (D) plants, so we can presume that we prolonged mainly the period from senescence onset to cotyledon abscission in D plants. In UV C irradiated plants the antioxidative enzymes were probably destroyed before the process of senescence could begin.     

Changes in Antioxidative Protection in Bean Cotyledons During Natural and Continuous Irradiation-Accelerated Senescence

We employed continuous irradiation (CL) for induction of premature senescence caused by enhanced production of reactive oxygen species. As a model plant we used bean (Phaseolus vulgaris L. cv. Jantar) cotyledons because they have well defined and a quite short life span. Senescence of bean cotyledons induced by CL progressed more rapidly than natural senescence: the life span of CL cotyledons was 13 d compared to 16 d in controls (C). Chl content was significantly lower in 10- and 13-d-old CL plants than in C plants and the change with age was not statistically significant. Activities of all antioxidative enzymes declined either with senescence onset or during whole life span. Activity of antioxidative enzymes, except ascorbate peroxidase, was lower in CL plants compared to C plants. On the contrary, contents of non-enzymatic antioxidants -carotene and ascorbate were higher in CL plants than in C plants. No significant difference, except in the youngest cotyledons, was observed in glutathione content.     

The capacity of antioxidant protection during modulated ageing of bean (Phaseolus vulgaris L.) cotyledons. 2. The low-molecular weight antioxidants

The aim of the present work was to evaluate non-enzymic antioxidants during natural and artificially modulated senescence. Senescence of bean (Phaseolus vulgaris L. cv. Jantar) cotyledons was modulated by UV C irradiation or by the decapitation of plants apices. The content of beta-carotene and zeaxanthin decreased in control and decapitated plants but in UV C irradiated plants these contents increased. The degree of de-epoxidation increased in all cultivations with age. The content of total glutathione (sum of reduced and oxidized) sharply decreased in bean cotyledons grown in all conditions. Interestingly, the content of total ascorbate increased at the end of cotyledon life span of control and decapitated plants but decreased in UV plants. Decrease of reduced/oxidized ratio of ascorbate and glutathione during cotyledon ageing confirmed increasing oxidative stress during senescence in all cultivations.     

Changes in Ultrastructure of Phaseolus Vulgaris L. Cotyledons Associated with their Modulated Life Span

French bean (Phaseolus vulgaris L.) cotyledons lost most of their reserve substances during several early days of germination and turned green. In cotyledon mesophyll cells of one-week-old seedlings, plastids were represented predominantly by amyloplasts (starch grains) and chloroamyloplasts, and the cells appeared to be metabolically highly active. Cell heterogeneity associated with distance of the cells from cotyledon vascular bundles was evident. Only mesophyll cells near to the bundles were rich in plastids. In two-weeks-old intact bean plants, the cotyledons were yellow and shrunken, and their cells were nearly "empty". The plastids in them were represented by senescent plastids (gerontoplasts) only. In the gerontoplasts as well as freely in cytosol, fluorescent lipoid inclusions were accumulated. This cotyledon development was more or less independent of irradiance. In "decapitated" bean plants, senescence of mesophyll cells and plastids was slowed down considerably, and the life span of the cotyledons was prolonged.     

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。     

Changes in endogenous cytokinin levels in cotyledons of Cucurbita pepo (zucchini) during natural and dark-induced senescence

Cytokinin (CK) levels in cotyledons of Cucurbita pepo L. (zucchini) were investigated through the processes of post-germination, greening, natural senescence and subsequent rejuvenation. The concentrations of the physiologically active CK bases, ribosides and nucleotides, as well as the cis -isomers of zeatin derivatives, decreased between the first and fifth weeks of cultivation under controlled light conditions. At the same time, the levels of storage CK O -glucosides and physiologically inactive CK 7- and 9-glucosides increased with senescence. With plant decapitation and subsequent cotyledon rejuvenation, not only the chlorophyll content but also the levels of physiologically active CKs, nucleotides and cis -zeatin derivatives increased. The levels of O -glucosides, however, decreased. When 1-week-old seedlings were transferred to the dark, there was a progressive reduction in cotyledon chlorophyll content, deterioration of chloroplast ultrastructure and a decrease in physiologically active CKs and their nucleotides. In contrast with natural senescence, the storage CK O -glucosides decreased under dark conditions, suggesting different metabolic regulation of endogenous CK levels during natural and dark-induced senescence of zucchini cotyledons. The chlorophyll loss of dark-treated cotyledons could be partially reversed, even after 5 days, with return to light conditions. During this recovery, physiologically active CKs and their nucleotides again increased, whereas the storage CK O -glucosides and cis -zeatins decreased. The present results suggest that dark-induced destruction and subsequent restoration of chloroplasts during light shifts are controlled by changes in the levels of physiologically active CKs and their nucleotides.     

Identification of nucleases related to nutrient mobilization in senescing cotyledons from French bean

The knowledge about the physiological function of plant nucleases is scarce besides that they have been involved in nucleic acid degradation related with programmed cell death processes. Cotyledons provide a suitable system to investigate this process and the changes associated to nutrient mobilization. Nuclease activities have been determined in French bean seedlings. The total nuclease activity in French bean cotyledons is lower than in embryonic axes; however, several nucleases were detected by in-gel nuclease activity assays with extracts from cotyledons of French bean and ssDNA as substrate. The nuclease activities induced during cotyledon senescence showed higher activity at neutral than at acidic pH. Five different nuclease genes belonging to S1/P1 family have been identified in French bean genome database named PVN1 to PVN5. Their relative expression in cotyledons has been determined from the start of imbibition to senescence, and three genes from this family showed expression in cotyledons. PVN1 was expressed during early stages of seedlings development, whereas PVN4 and PVN5 were expressed during cotyledons senescence. The removal of epicotyl in French bean seedlings resulted in a decrease in the activity and in the expression of the genes associated with the cotyledons senescence process, i.e. PVN4 and PVN5. At the same time, the mobilization of reserves in those cotyledons was slowed down. In the same way, the deficit in phosphate and nitrate during seedlings development led to an acceleration of induction of these genes at the same time that reserves were utilized early on the time. Therefore, the induction of PVN4 and PVN5, the two S1 nuclease genes involved in the process of cotyledon senescence, is related to nutrient mobilization, supporting a possible role for nucleic acids in nutrient recycling during cotyledon senescence.     

Different effects of dark treatment on pigment composition and photosystem I and II activities in intact cotyledons and primary leaves of Cucurbita pepo (zucchini)

Darkness mediates different senescence-related responses depending on the targeting of dark treatment (whole plants or individual leaves) and the organs that perceive the signal (leaves or cotyledons). In this study, we presented data on the differential effects of 2-day dark treatment on progression of senescence in cotyledons and primary leaves of 14-day-old plants of Cucurbita pepo L. (zucchini). The lack of changes in chlorophyll a, chlorophyll b and carotenoid contents as well as in the PSI activity measured by the far-red induced alterations in the P₇₀₀ oxidation levels and the quantum yield of electron transport from Q _A ⁻ to PSI end acceptors () indicated higher resistance of cotyledons to the applied dark stress compared to the primary leaves. In contrast to cotyledons, PSI activity in the primary leaves was significantly inhibited. Concerning the activity of PSII analyzed by the changes in the JIP-test parameters (the maximal efficiency of PSII photochemistry, ; the performance index, PI_ABS; the efficiency of Q _A ⁻ reoxidation, ψ ₀ and the effective dissipation per reaction center, DI₀/RC), no differences were observed between cotyledons and primary leaves, thus suggesting that PSI activity in the true leaves was more susceptible to the applied dark stress. The transfer of the darkened plants to normal light regime resulted in delayed senescence in cotyledons which was in contrast to results on Arabidopsis, thus implying the existence of specific mechanisms of cotyledon senescence in different monocarpic plants.     

The Role of Cytokinins and Ethylene in Bean Cotyledon Senescence. The Effect of Free Radicals

During ageing of bean (Phaseolus vulgaris L.) cotyledons in plants with modified life span the time-course of four cytokinins, ethylene, and the end products of free radical attack, lipofuscin-like pigments (LFP), were studied. UV irradiation shortened cotyledon life span, while epicotyl decapitation prolonged it. In controls, LFP increased at the senescence onset but at the end of life span it returned to the initial level. Ethylene increased more than 3-fold at the time of abscission. The content of individual cytokinins (zeatin, zeatin riboside, isopentenyl adenine, isopentenyl adenine riboside) varied differently during ageing but they did not decreased in any case under level observed in young cotyledons at the time of abscission. UV irradiation resulted in 14-fold increase in LFP concentration at the end. Ethylene increased 8-fold 2 h after irradiation. Individual cytokinins increased after UV irradiation to a different extent and time-course, nevertheless cotyledon life span was shortened. Decapitation induced LFP decrease. On day 13, LFP abruptly increased and than decreased and stayed lowered until abscission. Ethylene was maximum on day 24, at the time of abscission, it was above 200 % of control. Decapitation produced transient decrease in some cytokinins namely zeatin and isopentenyl adenine riboside.     

Studies on energy status and mitochondrial respiration during growth and senescence of mung bean cotyledons

Several characteristics of mitochondrial respiration and energy status have been studied during growth and senescence of mung bean ( Phaseolus radiatus L.) cotyledons. The results showed that mitochondrial oxygen consumption, respiratory control, ADP:O ratios, and energy charge changed in the cotyledons during germination and growth of the seedlings. The respiration rate of intact cotyledons approximately reflected the trend of the oxidative activities of the isolated mitochondria. An increase was observed in both whole cotyledon respiration and mitochondrial oxygen uptake at the onset of senescence of mung bean cotyledons (day 3 after germination), which thereafter declined gradually. The capacity and activity of the alternative pathway increased markedly in mitochondria isolated from senescent cotyledons. After the onset of senescence, the mung bean cotyledon mitochondria exhibited a decrease both in the respiratory control ratios and ADP:O ratios, and the cotyledons exhibited a gradual decline in energy charge. All these results showed an irreversible deterioration of energy conservation in mung bean cotyledons. The role(s) of the alternative pathway in senescent mung bean cotyledons is discussed.     

萌发绿豆子叶衰老期间的呼吸作用与线粒体功能的改变

暗中培养的绿豆幼苗子叶在萌发后3—4天时,外观出现衰老征状,6天后子叶凋落。随子叶日龄的增加,子叶的呼吸强度一直下降,呼吸商始终小于1。当外加L—苹果酸、a—酮戊二酸、琥珀酸和NADH为底物测定离体线粒体氧化活性时,衰老子叶的线粒体对上述四种底物的氧化活性有不同程度的增加;抗氰呼吸也有所升高。子叶衰老时,线粒体的ADP／O和呼吸控制(RC值均降低);线粒体ATPase水解ATP的活性升高。衰老绿豆子叶线粒体氧化磷酸化偶联效率的降低和ATPase水解活性的增强是与线粒体结构改变相联系的一种功能变化,它导致能量亏缺,并进一步加速了衰老的恶化进程。     

Senescence progression in a single darkened cotyledon depends on the light status of the other cotyledon in Cucurbita pepo (zucchini) seedlings: potential involvement of cytokinins and cytokinin oxidase/dehydrogenase activity

Darkness mediates different senescence-related responses depending on the targeting of dark treatment (whole plants or individual leaves) and on the organs that perceive the signal (leaves or cotyledons). As no data are available on the potential role of darkness to promote senescence when applied to individual cotyledons, we have investigated how darkness affects the progression of senescence in either a single or both individually darkened cotyledons of young 10-day-old Cucurbita pepo (zucchini) seedlings. Strong acceleration of senescence was observed when both cotyledons were darkened as judged by the damage in their anatomical structure, deterioration of chloroplast ultrastructure in parallel with decreased photosynthetic rate and photochemical quantum efficiency of PSII. In addition, the endogenous levels of cytokinins (CKs) and IAA were strongly reduced. In a single individually darkened cotyledon, the structure and function of the photosynthetic apparatus as well as the contents of endogenous CKs and IAA were much less affected by darkness, thus suggesting inhibitory effect of the illuminated cotyledon on the senescence of the darkened one. Apparently, the effect of darkness to accelerate/delay senescence in a single darkened cotyledon depends on the light status of the other cotyledon from the pair. The close positive correlation between CK content and the activity of CK oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) suggested that CKX was essentially involved in the mechanisms of downregulation of endogenous CK levels. Our results indicated that CKX-regulated CK signaling could be a possible regulatory mechanism controlling senescence in individually darkened cotyledons.     

Induction characteristics and response of photosynthetic quantum conversion to changes in irradiance in mulberry plants

A study was conducted, using rapid time course of chlorophyll (Chl) fluorescence parameters, and light-response curves of Chl fluorescence parameters, to determine the induction requirements and response of photosystem II (PSII) photochemistry and non-photochemical reactions after changes in irradiance in greenhouse mulberry plants. The induction of PSII photochemistry rapidly approached to steady state after leaves were treated from darkness to low irradiance (LI). When irradiance of leaves changed from darkness to high irradiance (HI), a biphasic induction was observed. A slight photoinhibition occurred in the leaves exposed to sunlight coming to the greenhouse, whereas a chronic photoinhibition occurred in the leaves fully exposed to sunlight outside the greenhouse. The chronic photoinhibition was demonstrated by sustained reduction of maximal quantum yield of PSII photochemistry (Fv/Fm). Moreover, the leaves of mulberry plants in greenhouse were sensitive to abrupt changes in irradiance and the sensitivity of leaves suffered in a short-term (1h) high light treatment was reduced, based on the changes in photosynthetic quantum conversion. These results demonstrated an inducible response of photosynthetic quantum conversion to changes in irradiance in mulberry.     

Enzymatic changes in gourd and bean cotyledons during ageing and the effect of detopping

Cotyledons of gourd (Cucurbita maxima Duchesne) and bean (Phaseolus vulgaris L.) were used to study the changes in the activities of catalase, peroxidase, acid inorganic pyrophosphatase and alkaline inorganic pyrophosphatase during ageing and the diversion in such changes that occur when cotyledon senescence was retarded by detopping the seedlings above the cotyledons. Catalase, acid inorganic pyrophosphatase and alkaline inorganic pyrophosphatase activities declined during the senescence of the cotyledons. When cotyledon senescence was retarded by detopping as marked by the increase in the levels of chlorophyll and protein, there was also an increase in the activities of these enzymes. Peroxidase activity, on the other hand, increased during the senescence of the cotyledons and detopping the seedlings resulted in a further increase in the peroxidase activity. It can be suggested that some root factor(s) probably cytokinin(s) is (are) mobilised into the cotyledons of the detopped seedlings which otherwise would have been mobilised into the shoot apices, and help retard or even reverse the senescence of the cotyledons.     

Photoinhibition of Photosynthesis: Role of Carotenoids in Photoprotection of Chloroplast Constituents

Exposure of plants to irradiation, in excess to saturate photosynthesis, leads to reduction in photosynthetic capacity without any change in bulk pigment content. This effect is known as photoinhibition. Photoinhibition is followed by destruction of carotenoids (Cars), bleaching of chlorophylls (Chls), and increased lipid peroxidation due to formation of reactive oxygen species if the excess irradiance exposure continues. Photoinhibition of photosystem 2 (PS2) in vivo is often a photoprotective strategy rather than a damaging process. For sustainable maintenance of chloroplast function under high irradiance, the plants develop various photoprotective strategies. Cars perform essential photoprotective roles in chloroplasts by quenching the triplet Chl and scavenging singlet oxygen and other reactive oxygen species. Recently photoprotective role of xanthophylls (zeaxanthin) for dissipation of excess excitation energy under irradiance stress has been emphasised. The inter-conversion of violaxanthin (Vx) into zeaxanthin (Zx) in the light-harvesting complexes (LHC) serves to regulate photon harvesting and subsequent energy dissipation. De-epoxidation of Vx to Zx leads to changes in structure and properties of these xanthophylls which brings about significant structural changes in the LHC complex. This ultimately results in (1) direct quenching of Chl fluorescence by singlet-singlet energy transfer from Chl to Zx, (2) trans-thylakoid membrane mediated, pH-dependent indirect quenching of Chl fluorescence. Apart from these, other processes such as early light-inducible proteins, D1 turnover, and several enzymatic defence mechanisms, operate in the chloroplasts, either for tolerance or to neutralise the harmful effect of high irradiance.     

Study of the senescence process in primary leaves of sunflower (Helianthus annuus L.) plants under two different light intensities

Different parameters that vary during leaf development may be affected by light intensity. To study the influence of different light intensities on primary leaf senescence, sunflower (Helianthus annuus L.) plants were grown for 50 days under two photon flux density (PFD) conditions, namely high irradiance (HI) at 350 μmol(photon) m^?2 s^?1 and low irradiance (LI) at 125 μmol(photon) m^?2 s^?1. Plants grown under HI exhibited greater specific leaf mass referred to dry mass, leaf area and soluble protein at the beginning of the leaf development. This might have resulted from the increased CO₂ fixation rate observed in HI plants, during early development of primary leaves. Chlorophyll a and b contents in HI plants were lower than in LI plants in young leaves. By contrast, the carotenoid content was significantly higher in HI plants. Glucose concentration increased with the leaf age in both treatments (HI and LI), while the starch content decreased sharply in HI plants, but only slightly in LI plants. Glucose contents were higher in HI plants than in LI plants; the differences were statistically significant (p<0.05) mainly at the beginning of the leaf senescence. On the other hand, starch contents were higher in HI plants than in LI plants, throughout the whole leaf development period. Nitrate reductase (NR) activity decreased with leaf ageing in both treatments. However, the NR activation state was higher during early leaf development and decreased more markedly in senescent leaves in plants grown under HI. GS activity also decreased during sunflower leaf ageing under both PFD conditions, but HI plants showed higher GS activities than LI plants. Aminating and deaminating activities of glutamate dehydrogenase (GDH) peaked at 50 days (senescent leaves). GDH deaminating activity increased 5-fold during the leaf development in HI plants, but only 2-fold in LI plants. The plants grown under HI exhibited considerable oxidative stress in vivo during the leaf senescence, as revealed by the substantial H₂O₂ accumulation and the sharply decrease in the antioxidant enzymes, catalase and ascorbate peroxidase, in comparison with LI plants. Probably, systemic signals triggered by a high PFD caused early senescence and diminished oxidative protection in primary leaves of sunflower plants as a result.     

Effects of low irradiation on photosynthesis and antioxidant enzyme activities in cucumber during ripening stage

In order to investigate the effects of low irradiation (LI) on cucumber (Cucumis sativus L. cv. Jinyou 35) during a ripening stage, our experiment was carried out in a climate chamber. Two levels of PAR were set for plants: normal irradiation [NI, 600 μmol(photon) m^?2 s^?1] and low irradiation [LI, 100 μmol(photon) m^?2 s^?1], respectively. The experiments lasted for 9 d; then both groups of plants were transferred under NI to recover for 16 d. The plants showed severe chlorosis after the LI treatment. Chlorophyll (Chl) a, initial slope, photosynthetic rate at saturating irradiation (P_max), light saturation point, maximal photochemical efficiency of PSII (F_v/F_m), electron transport rate of PSII (ETR), soluble protein content, and catalase (CAT) activity in cucumber leaves decreased under LI stress, while Chl b, carotenoids, light compensation point, nonphotochemical quenching (q_N), superoxide dismutase (SOD), and malondialdehyde (MDA) exhibited an increasing trend under LI. After 16 d of recovery, values of P_max, F_v/F_m, ETR, q_N, SOD, CAT, MDA, and soluble protein were close to those of the control after one, three, and five days of the LI treatment, while those kept under LI for 7 and 9 d could not return to the control level. Therefore, 7 d of LI stress was a meteorological disaster index for LI in cucumber at the fruit stage.     

Methyl jasmonate-dependent senescence of cotyledons in <Emphasis Type="Italic">Ipomoea nil</Emphasis>

Jasmonic acid methyl ester (JAMe) has been recently shown to play a crucial role in many physiological processes. In this paper, we focused on cotyledon senescence in Ipomoea nil and revealed that JAMe and darkness are the main factors stimulating the process examined. What is more, we showed that mefenamic acid (a jasmonate biosynthesis inhibitor) reverses the stimulatory effect of darkness on senescence. In plants growing under dark conditions, stimulation of JASMONIC ACID CARBOXYL METHYLTRANSFERASE (InJMT) expression and, consequently, an increase in JAMe content, have been observed. In turn, the level of jasmonic acid (JA) gradually decreased. Moreover, dark-grown seedlings demonstrated a lower PSII functional activity and a reduced chlorophyll content and autofluorescence. All of these data suggest that JAMe is a signal molecule controlling the senescence of cotyledons in I. nil.     

Differential changes in the steady state levels of thylakoid membrane proteins during senescence in Cucumis sativus cotyledons

Chloroplast structure and function is known to alter during foliar senescence. Besides, the alterations in the structural organisation of thylakoid membranes changes in the steady state levels of thylakoid membrane proteins occur due to leaf ageing. We monitored temporal changes in some of the specific proteins of thylakoid membrane protein complexes by western blotting in the Cucumis sativus cotyledons as a function of the cotyledon age. We observed that the levels of D1 and D2 proteins of photosystem II started declining at the early stages of senescence of Cucumis cotyledons and continued to decline with the progress of cotyledon age. Similarly the level of Cyt f of Cyt b6/f complex declined rapidly with progress of senescence in these cotyledons. The reaction centre proteins of photosystem I were relatively found to be more stable than that of photosystem II reaction centre proteins reflecting possibly the disorganisation of photosystem II prior to photosystem I. The 33 kDa extrinsic protein (MSP) of oxygen evolving complex, the LHCII apoprotein and the beta-subunit of ATPsynthase showed the declined levels with the progress of cotyledon age. However, the extents of loss of these proteins were not as high as the reaction centre proteins of photosystem II and the Cyt f. These results provide that during senescence, proteins of thylakoid membranes degrade in a specific temporal sequence and thereby affect the temporal photochemical functions in Cucumis sativus cotyledons.