Ultra-structural and functional changes in the chloroplasts of detached barley leaves senescing under dark and light conditions

Changes in the chloroplast ultra-structure and photochemical function were studied in detached barley (Hordeum vulgare L. cv. Akcent) leaf segments senescing in darkness or in continuous white light of moderate intensity (90 mumol m-2 s-1) for 5 days. A rate of senescence-induced chlorophyll degradation was similar in the dark- and light-senescing segments. The Chl a/b ratio was almost unchanged in the dark-senescing segments, whereas in the light-senescing segments an increase in this ratio was observed indicating a preferential degradation of light-harvesting complexes of photosystem II. A higher level of thylakoid disorganisation (especially of granal membranes) and a very high lipid peroxidation were observed in the light-senescing segments. In spite of these findings, both the maximal and actual photochemical quantum yields of the photosystem II were highly maintained in comparison with the dark-senescing segments.     

Changes in activity of energy dissipating mechanisms in wheat flag leaves during senescence

Abstract: Excitation energy dissipation, including the xanthophyll cycle, during senescence in wheat flag leaves grown in the field was investigated at midday and in the morning. With progress of senescence, photosynthesis (Pn) and actual PSII photochemical efficiency (ΦPSII) decreased markedly at midday. The decrease in extent of Pn was greater than that of ΦPSII. However, there was no significant decline in Pn and ΦPSII observed in the morning, except in leaves 60 days after anthesis. The kinetics of xanthophyll cycle activity, thermal dissipation (NPQ), and q_f observed at midday during senescence exhibited two distinct phases. The first phase was characterized by an increase of xanthophyll cycle activity, NPQ, and q_f during the first 45 days after anthesis. The second phase took place 45 days after anthesis, characterized by a dramatic decline in the above parameters. However, the qI, observed both at midday and in the morning, always increased along with senescence. A larger proportion of NPQ insensitive to DTT (an inhibitor of the de-epoxidation of V to Z) was also observed in severely senescent leaves. In the morning, only severely senescent leaves showed higher xanthophyll cycle activity, NPQ, q_f, and qI. It was demonstrated that, at the beginning of senescence or under low light, wheat leaves were able to dissipate excess light energy via NPQ, depending on the xanthophyll cycle. However, the xanthophyll cycle was insufficient to protect leaves against photodamage under high light, when leaves became severely senescent. The ratio of (Fj - Fo)/(Fp - Fo) increased gradually during the first 45 days after anthesis, but dramatically increased 45 days after anthesis. We propose that another photoprotection mechanism might exist around reaction centres, activated in severely senescent leaves to protect leaves from photodamage.     

Xanthophyll cycle activity in detached barley leaves senescing under dark and light

Senescence-induced changes in the xanthophyll cycle activity and chlorophyll (Chl) fluorescence parameters were compared in detached barley (Hordeum vulgare L.) leaf segments kept for 6 d in darkness or under continuous white light (90 mol m^–2 s^–1). Before detachment of the leaf segments, the plants were grown at periodic regime [12 h light (90 mol m^–2 s^–1)/12 h dark]. The de-epoxidation state of the xanthophyll cycle pigments (DEPS) in the leaf samples was determined immediately (the actual DEPS), after 1 h of dark-adaptation (the residual DEPS), and during 14 min of a high-irradiance (HI) exposure (500 mol m^–2 s^–1) (HI-induced DEPS). In the light-senescing segments, senescence was delayed pronouncedly compared to dark-senescing ones as the Chl content, the photosystem 2 photochemistry, and electron transport processes were highly maintained. Further, the actual DEPS increased, probably due to the increased mean photon dose. The HI-induced increase in the DEPS was stimulated in the light-senescing segments, whereas it was slowed down in the dark-senescing ones. However, after the 14 min HI-exposure of the dark-senescing segments the HI-induced DEPS was not markedly lower than in the mature leaves, which indicated the maintenance of the xanthophyll cycle operation.     

Influence of cytokinins and novel cytokinin antagonists on the senescence of detached leaves of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Cytokinins N⁶-benzyladenine (BA) and 1-(2-chloropyridin-4-yl)-3-phenylurea (4PU-30) delayed the senescence of detached leaves (3^rd to 7^th leaf node) of wild and ethylene insensitive eti5 mutant of Arabidopsis thaliana. The novel anticytokinins, structural analogues of purine and phenylurea cytokinins also affected the senescence of detached rosette leaves of A. thaliana. They diminished to a significant extent the cytokinin-induced delay of chlorophyll destruction, but without a considerable difference in their action against both types of cytokinins. These results correlated with changes observed in ribonuclease (RNase) activity.     

Senescing leaves possess potential for stress adaptation: the developing leaves acclimated to high light exhibit increased tolerance to osmotic stress during senescence

Plants may experience environmental stress factors operating in nature either simultaneously or in sequence. In the study, we have acclimated the developing primary leaves of wheat seedlings to high light stress and examined their photosynthetic response to polyethylene glycol (PEG) mediated osmotic stress during different developmental phases including senescence. The high light acclimated leaves show higher level of total carotenoids as compared to their non-acclimated counterparts experiencing osmotic stress during senescence. They also exhibit greater membrane stability as indicated by the measurements of fluorescence polarisation and energy transfer efficiency in photosystem I (PSI) and Photosystem II (PSII). From the data of DCPIP photoreduction and pulse amplitude modulated (PAM) fluorimetry, a similar trend is observed for PSII photochemistry of the leaves experiencing osmotic stress during senescence. Our results may suggest that the stress adaptive potential induced by one stress during development is retained by the leaves and helps to mitigate another stress effect operating in sequence during another developmental phase, namely senescence.     

Changes in photosystem II function during senescence of wheat leaves

Analyses of chlorophyll fluorescence were undertaken to investigate the alterations in photosystem II (PSII) function during senescence of wheat ( Triticum aestivum L. cv. Shannong 229) leaves. Senescence resulted in a decrease in the apparent quantum yield of photosynthesis and the maximal CO₂ assimilation capacity. Analyses of fluorescence quenching under steady‐state photosynthesis showed that senescence also resulted in a significant decrease in the efficiency of excitation energy capture by open PSII reaction centers (F'_v/F'_m) but only a slight decrease in the maximum efficiency of PSII photochemistry (F'_v/F'_m). At the same time, a significant increase in non‐photochemical quenching (q_N) and a considerable decrease in photochemical quenching (q_P) were observed in senescing leaves. Rapid fluorescence induction kinetics indicated a decrease in the rate of Q_A reduction and an increase in the proportion of Q_B‐non‐reducing PSII reaction during senescence. The decrease in both F'_v/F'_m and q_P explained the decrease in the actual quantum yield of PSII electron transport ((φ_PSII). We suggest that the modifications in PSII function, which led to the down‐regulation of photosynthetic electron transport, would be in concert with the lower demand for ATP and NADPH in the Calvin cycle which is often inhibited in senescing leaves.     

Estimation of the light distribution between photosystems I and II in intact wheat leaves by fluorescence and photoacoustic measurements

Usisng intact leaves, the extent of the decrease in chlorophyll a fluorescenece caused by the addition of continuous 710 nm light superimposed on modulated (20 Hz) 550 nm light was used to determine the distribution of this absorbed light between photosystems I () and II (). The F_o and F_m levels, which defined the total variable fluorescenece, were taken as equal to those obtained with excess 710 nm light and with saturating blue-green light, respectively.An analogous procedure was used with a photoacoustic detector, saturating white light defining a base line for oxygen yield, the levels with an without 710 nm light being used to define and respectively.The two methods gave similar values for the distribution of light between the two photosystems for the experimental conditions used, averaging 0.55 for a range of Triticum genotypes and Brachypodium sylvaticum grown in high or low light.     

Paclobutrazol delayed dark-induced senescence of mung bean leaves

Sixty days old mung beans Vigna radiata (L.) Wilczek were treated with soil applied paclobutrazol, at the rate of 500 μg per 10 inch pot. After seven days of application, the plants along with untreated controls were transferred to the dark for induction of senescence. The treated plants exhibited higher chlorophyll content and activity of catalase (CAT) compared to controls. In contrast, control leaves had higher activity of peroxidase (POX) and a higher content of malondialdehyde (MDA), while superoxide dismutase (SOD) activity remained unchanged. Upon transfer to dark, chlorophyll content declined in both control and treated plants but the decline was much faster in control. The activity of CAT decreased significantly in controls while POX activity and MDA content remained higher in control than in treated plants. Paclobutrazol delayed the dark-induced senescence in attached mung bean leaves in association with the maintenance of higher activity of CAT, low activity of POX, and low MDA contents. The variation in SOD activity was not discernible with senescence levels.     

Protective cytokinin action switches to damaging during senescence of detached wheat leaves in continuous light

The effect of exogenous application of the cytokinin meta -topolin [mT; N⁶-( meta -hydroxybenzyl)adenine] on artificial senescence of detached wheat leaves ( Triticum aestivum L. cv. Hereward) was studied and compared in leaves senescing under continuous light (100 µmol photons m⁻² s⁻¹) and darkness. Senescence-induced deterioration in structure and function of the photosynthetic apparatus was characterized by reduction in chlorophyll content, maximal efficiency of photosystem (PS) II photochemistry ( F _v/ F _m) and the rate of CO₂ assimilation, by increase in the excitation pressure on PSII (1 −  q _P) and a level of lipid peroxidation and by modifications in chloroplast ultrastructure. While in darkened leaf segments mT effectively slowed senescence-induced changes in all measured parameters, in light-senescing segments the effect of mT changed into opposite a few days after detachment. We observed an overexcitation of photosynthetic apparatus, as indicated by pronounced increases in the excitation pressure on PSII and in a deepoxidation state of xanthophyll cycle pigments, marked starch grain accumulation in chloroplasts and stimulation of lipid peroxidation in light-senescing leaf segments in mT. Possible mechanisms of acceleration of senescence-accompanying decrease in photosynthetic function and increase in lipid peroxidation during mT influence are discussed. We propose that protective mT action in darkness becomes damaging during artificial senescence in continuous light due to overexcitation of photosynthetic apparatus resulting in oxidative damage.     

Reversible photoinactivation of photosystem II during desiccation of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L. cv. Albori) leaves in the light

We investigated the inherent protective mechanisms against stress and damage to photosystem II (PSII) in barley (Hordeum vulgare L. cv. Albori). Leaves were desiccated at 30% relative humidity, under either low or high light (photon flux densities of 100 or 300 μmol m^-2 s^-1, respectively). During the treatment period, relative water content dropped to 35 to 45%, depending on light intensity. However, the photochemical efficiency of PSII (Fv/Fm) decreased only about 10%. This relatively stable response was due to the rapid, reversible increase in Fm (maximum fluorescence) during 20 min of dark-adaptation. During desiccation in the light, however, PSII was photo-inactivated by non-photochemical quenching (NPQ), with the excess excitation energy absorbed by the chlorophyll being dissipated as heat energy. This decline in NPQ in the first 2 min of treatment was caused by a relaxation in the energy-dependent quenching during dark-adaptation, but could be delayed significantly by a phosphatase inhibitor, NaF. In addition, the relaxation of other NPQ components related to state transition and phosphorylation of thylakoid phosphoproteins were blocked by NaF.     

Effect of wounding on ethylene biosynthesis and senescence of detached spinach leaves

Parameters of senescence and ethylene biosynthesis pathway were screened simultaneously in detached spinach leaves and leaf discs. Senescence was enhanced by application of 1-aminocyclopropane-1-carboxylic acid (ACC) and was retarded by amino-ethoxyvinylglycine (AVG). Evidence is presented showing that the bursts of both wound- and climacteric-like ethylene promoted senescence of detached leaves and leaf discs. This ethylene-enhanced leaf senescence was dependent on: (a) ethylene production rates in the tissue; (b) the degree of wounding. Wounding resulted in elevated levels of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), which declined in advanced stages of senescence. The results suggest that wounding might be regarded as one of the primary events in the induction of the senescence syndrome in detached leaves and leaf discs, while ethylene is implicated as a regulator of the rate of the process.     

Non-photochemical quenching of Fo in leaves is emission wavelength dependent: consequences for quenching analysis and its interpretation

The effects of light-induced non-photochemical quenching on the minimal F_o, and variable F_v, fluorescence emissions at 690 and 730 nm in leaves were determined. Non-photochemical quenching of F_o, but not F_v, was found to be dependent upon the wavelength of emission, and was greater at 690 nm than at 730 nm. For emission at 730, compared to at 690 nm, approx. 30% of F_o was not affected by non-photochemical quenching processes in leaves of C3 plants; in maize leaves this was found to be approx. 50%. The data indicate that a substantial proportion of the pigments contributing to F_o emission at 730 nm are not quenched by light-induced, non-photochemical quenching processes and that there are large differences in the pigment matrices contributing to F_o and F_v emissions at 730 nm, compared to those at 690 nm. These findings have important implications for the accurate estimation and interpretation of non-photochemical quenching of fluorescence parameters and their use in the calculation of photochemical efficiencies in leaves. Measurements of fluorescence emissions at wavelengths above 700 nm are likely to give rise to significant errors when used for determinations of photochemical and non-photochemical quenching parameters.     

Enzymes of nitrogen mobilization in detached leaves of Lolium temulentum during senescence

During the senescence of Lolium temulentum leaf sections in the dark, asparagine and glutamine accumulated as the level of soluble protein declined. During the first 3–4 days after detachment, when the rate of protein loss was maximal, a four-fold increase in acid protease activity (EC 3.4.4.?) occurred. Subsequently this activity was replaced by proteases with a higher pH optimum. There was also a pronounced and continued activation of glutamate dehydrogenase (EC 1.4.1.2) during senescence. Glutamate pyruvate transaminase (EC 2.6.1.2), benzoylarginine-p-nitroanilide hydrolase (EC 3.4.?.?) and leucyl-p-nitroanilide hydrolase (EC 3.4.1.1) declined from high initial activities after 3–4 days. Glutamate oxaloacetate transaminase (GOT, EC 2.6.1.1) was fairly stable although a marked increase occurred in the activity of one of two major GOT isoenzymes over the first two days. Glutamine synthetase (EC 6.3.1.2) was highly active in non-senescent leaves but fell sharply during the first three days of senescence. Little asparagine synthetase (EC 6.3.1.1) was detected. The role of these enzymes in the nitrogen metabolism of senescent detached leaves is discussed.     

The effects of light,cycloheximide and incubation medium on the senescence of detached seagrass leaves

The senescence of detached leaves of the Australian seagrassZostera muelleri Irmisch ex Aschers was associated with decreased levels of chlorophyll, soluble protein, and alkaline pyrophosphatase and catalase activities. Senescence was faster in artificial seawater than in distilled water. Irradiance has a retarding effect on senescence while cycloheximide did not retard senescence appreciably. The action of cycloheximide was studied usingCyperus rotundus L.,Hydrangea macrophylla (Thunb.) Ser.,Solandra maxima (Sesśe & Moc.) P. S. Green, andDatura suaveolens Humb. & Bonpl., and the cycloheximide sensitivity correlated with low chlorophyllase activity of the leaf. An erratum to this article is available at .     

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.     

The post-illumination chlorophyll fluorescence transient indicates the RuBP regeneration limitation of photosynthesis in low light in Arabidopsis

The mechanism of post-illumination chlorophyll fluorescence transient (PIFT) was investigated in Arabidopsis. PIFT was detected in the wild type after illumination with low light. In the fba3-2 (fructose-1,6-bisphosphate aldolase) mutant, in which PIFT is enhanced, strong light also induced PIFT. PIFT was suppressed not only in the triose phosphate/phosphate translocator (tpt-2) mutant, but also in tpt-2 fba3-2, suggesting that triose phosphates, such as dihydroxyacetone phosphate (DHAP), are involved in the PIFT mechanism. We concluded that PIFT is associated with ribulose-1,5-bisphosphate (RuBP)-regeneration limitation of photosynthesis in low light.     

Changes in leaf protein and pigment contents and photosynthetic activities during senescence of detached maize leaves: influence of different ultraviolet radiations

Senescence induced loss in pigments and proteins of detached maize (Zea mays L. cv. Col) leaves was significantly enhanced on the exposure of leaves to different ranges of ultraviolet (UV) radiation. Compared to UV-A (320-400 nm) and UV-B (280-320 nm), the UV-C (200-320 nm) was the most damaging for the pigments and macromolecules. A severe decline in photosystem (PS) 2 mediated photoreduction during senescence of detached leaves exposed to UV irradiation suggested a damage of the system. The PS1 mediated photoreduction of methylviologen with 2,6-dichlorophenol indophenol as electron donor was stimulated by UV-A and UV-B radiations, suggesting a reorganisation of the PS1 complex. These results were fortified by the values of fast and slow kinetics of chlorophyll (Chl) a fluorescence transients. This revised version was published online in June 2006 with corrections to the Cover Date.     

The light stress-induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana

The early light-induced proteins (ELIPs) belong to the multigenic family of pigment-binding light-harvesting complexes. ELIPs accumulate transiently and are believed to play a protective role in plants exposed to high levels of light. Constitutive expression of the ELIP2 gene in Arabidopsis resulted in a marked reduction of the pigment content of the chloroplasts, both in mature leaves and during greening of etiolated seedlings. The chlorophyll loss was associated with a decrease in the number of photosystems in the thylakoid membranes, but the photosystems present were fully assembled and functional. A detailed analysis of the chlorophyll-synthesizing pathway indicated that ELIP2 accumulation downregulated the level and activity of two important regulatory steps: 5-aminolevulinate synthesis and Mg-protoporphyrin IX (Mg-Proto IX) chelatase activity. The contents of glutamyl tRNA reductase and Mg chelatase subunits CHLH and CHLI were lowered in response to ELIP2 accumulation. In contrast, ferrochelatase activity was not affected and the inhibition of Heme synthesis was null or very moderate. As a result of reduced metabolic flow from 5-aminolevulinic acid, the steady state levels of various chlorophyll precursors (from protoporphyrin IX to protochlorophyllide) were strongly reduced in the ELIP2 overexpressors. Taken together, our results indicate that the physiological function of ELIPs could be related to the regulation of chlorophyll concentration in thylakoids. This seems to occur through an inhibition of the entire chlorophyll biosynthesis pathway from the initial precursor of tetrapyrroles, 5-aminolevulinic acid. We suggest that ELIPs work as chlorophyll sensors that modulate chlorophyll synthesis to prevent accumulation of free chlorophyll, and hence prevent photooxidative stress.     

Different strategies of Cd tolerance and accumulation in Arabidopsis halleri and Arabidopsis arenosa

Pseudometallophytes are commonly used to study the evolution of metal tolerance and accumulation traits in plants. Within the Arabidopsis genus, the adaptation of Arabidopsis halleri to metalliferous soils has been widely studied, which is not the case for the closely related species Arabidopsis arenosa. We performed an in-depth physiological comparison between the A. halleri and A. arenosa populations from the same polluted site, together with the geographically close non-metallicolous (NM) populations of both species. The ionomes, growth, photosynthetic parameters and pigment content were characterized in the plants that were growing on their native site and in a hydroponic culture under Cd treatments. In situ, the metallicolous (M) populations of both species hyperaccumulated Cd and Zn. The NM population of A. halleri hyperaccumulated Cd and Zn while the NM A. arenosa did not. In the hydroponic experiments, the NM populations of both species accumulated more Cd in their shoots than the M populations. Our research suggests that the two Arabidopsis species evolved different strategies of adaptation to extreme metallic environments that involve fine regulation of metal homeostasis, adjustment of the photosynthetic apparatus and accumulation of flavonols and anthocyanins.     

The different fates of mitochondria and chloroplasts during dark-induced senescence in Arabidopsis leaves

Senescence is an active process allowing the reallocation of valuable nutrients from the senescing organ towards storage and/or growing tissues. Using Arabidopsis thaliana leaves from both whole darkened plants (DPs) and individually darkened leaves (IDLs), we investigated the fate of mitochondria and chloroplasts during dark-induced leaf senescence. Combining in vivo visualization of fates of the two organelles by three-dimensional reconstructions of abaxial parts of leaves with functional measurements of photosynthesis and respiration, we showed that the two experimental systems displayed major differences during 6 d of dark treatment. In whole DPs, organelles were largely retained in both epidermal and mesophyll cells. However, while the photosynthetic capacity was maintained, the capacity of mitochondrial respiration decreased. In contrast, IDLs showed a rapid decline in photosynthetic capacity while maintaining a high capacity for mitochondrial respiration throughout the treatment. In addition, we noticed an unequal degradation of organelles in the different cell types of the senescing leaf. From these data, we suggest that metabolism in leaves of the whole DPs enters a 'stand-by mode' to preserve the photosynthetic machinery for as long as possible. However, in IDLs, mitochondria actively provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. Finally, the heterogeneity of the degradation processes involved during senescence is discussed with regard to the fate of mitochondria and chloroplasts in the different cell types.