Molecular analysis of a phytohemagglutinin-defective cultivar of Phaseolus vulgaris L.

Plastoglobuli have been isolated and purified from chloroplasts of beech and spinach leaves and from broom flower chromoplasts by a repeated floating-gradient technique. The main components in plastoglobuli isolated from chloroplasts were triacylglycerols and lipophilic prenyl quinones, mainly plastohydroquinone and -tocopherol. The corresponding oxidized prenyl quinones, plastoquinone (ox), -tocoquinone, and the phylloquinone vitamin K₁, were detected in trace amounts. Plastoglobuli isolated from chromoplasts contained large amounts of carotenoid esters. Triacylglycerols constituted two-thirds of the content of these plastoglobuli. The total prenyl quinone content was low in chromoplast plastoglobuli. Plastoquinone (ox) was the major prenyl quinone constituent. Plastoglobuli contained small amounts of chlorophylls, carotenoids (with the exception of chromoplast plastoglobuli), glycolipids, and proteins due to adsorption phenomena during the isolation process; however, increasing purification of the plastoglobuli fractions resulted in an exponential decline of these components. Adsorption of thylakoid lipids onto the plastoglobuli during the isolation process was demonstrated using an artificial globuli system. Therefore, pigments, glyco- and phospholipids, and proteins were regarded as thylakoid contaminations and not as actual constituents of plastoglobuli.     

Accumulation of phytoene in plastoglobuli of SAN-9789 (Norflurazon)-treated dark-grown wheat

Grains of wheat ( Triticum aestivum L. cv. Starke II) were treated with SAN-9789 (Norflurazon), and grown in darkness for 6 days. The SAN treatment resulted in an inhibition of the carotenoid synthesis at the level of phytoene. The plastids of SAN-treated plants contained enlarged and non-osmiophilic plastoglobuli, compared to the plastoglobuli of the control. The plastoglobuli were isolated and purified by means of a flotation technique, and their lipid composition was determined. Efforts were made to avoid contamination of epicuticular soluble lipids in the plastoglobuli suspension during isolation. The most suitable method was found to be a mechanical removal of the lipids from the surface of the intact leaves prior to homogenization. Membrane lipids, i.e. galacto-, phospho- and sulpholipids could not be detected in plastoglobuli from either SAN-treated or control plants, indicating that contamination with membranes was negligible. In plastoglobuli of SAN-treated plants, large amounts of phytoene and, to a lesser extent, phytofluene accumulated. The proportion of triacylglycerols to quinones was lower than in the control. The main lipids in control plastoglobuli were triacylglycerols, plastoquinones and α-tocopherol. The possible function of plastoglobuli in etioplasts is discussed.     

Differential effects of benzyladenine on the ultrastructure of chloroplasts in intact bean leaves according to their age

Summary Primary leaves of intact bean plants (Phaseolus vulgaris) were treated with benzyladenine (BA) at different stages of growth. Changes in the ultrastructure of chloroplasts and the contents of chlorophyll, carotenoid, and protein (soluble and insoluble) in leaves with different treatments were followed and compared. When BA was applied from an early stage, it increased the chloroplast size and the number of grana per chloroplast without any pronounced effect on the grana size. When BA treatment was stopped at the early stage, these effects remained for a while and then diminished. When BA treatment was begun at a late stage, such marked effects were not observed, suggesting that only young leaves could respond to BA in that manner. However, the late treatment efficiently prevented the process of the last stage of leaf senescence characterized by disintegration of thylakoids with concomitant increase in the plastoglobule size. Chlorophyll, carotenoid, and insoluble protein contents per leaf followed similar changes in chloroplast length and the number of grana per chloroplast section.     

Tocopherol quinone content of green algae and higher plants revised by a new high-sensitive fluorescence detection method using HPLC--effects of high light stress and senescence

A rapid, sensitive fluorescence method was applied here for detection of oxidized tocopherol quinones in total plant tissue extracts using HPLC, employing a post-column reduction of these compounds by a Zn column. Using this method, we were able to detect both alpha- and gamma-tocopherol quinones in Chamydomonas reinhardii with a very high degree of sensitivity. The levels of both compounds increased under high light stress in the presence of pyrazolate in parallel to a decrease in the content of the corresponding tocopherols. The formation of tocopherol quinones from tocopherols was apparently due to their oxidation by singlet oxygen, which is formed in photosystem II under high light stress. alpha-Tocopherol quinone was also detected in a variety of higher plants of different age, and its level was found to increase during senescence in leaves grown under natural conditions. In contrast to alpha-tocopherol quinone, gamma-tocopherol quinone was not found in the higher plant species investigated with the exception of young runner bean leaves, where the levels of both compounds increased dramatically during cold and light stress. Taking advantage of native fluorescence of the reduced alpha-tocopherol quinone (alpha-tocopherol quinol), it can be detected in plant tissue extracts with a high sensitivity. In young runner bean leaves, alpha-tocopherol quinol was found at a level similar to alpha-tocopherol.     

Does the chloroplast envelope contain carotenoids and quinones in vivo?

Leaves, cotyledons, isolated chloroplasts and subplastid fractions (thylakoids and envelopes) of radish ( Raphanus sativus L. cv. Saxa) and spinach ( Spinacia oleracea L. cv. Matador) were assayed for their pigment and quinone content and composition. Evidence is presented suggesting that the chloroplast envelope does not contain carotenoids and quinones in vivo. Envelopes prepared by the method described contained very low amounts of chlorophyll a and b , violaxanthin and neoxanthin, but no β-carotene, lutein, zeaxanthin and antheraxanthin. Among the quinones, trace amounts of plastoquinone and α-tocopherol but no plastohydroquinone, α-tocoquinone and phylloquinone were detected. The data presented suggest that, contrary to previous findings, carotenoids and quinones are not located in the chloroplast envelope.     

Accumulation of Plastoquinone A during Low Temperature Growth of Winter Rye

Chloroplasts isolated from rye (Secale cereale L. cv Puma) grown at 5°C (RH) accumulated 260% more plastoquinone A (PQA) per plastid than chloroplasts isolated from rye grown at 20°C (RNH). The number of plastoglobuli increased by 270% in RH chloroplasts compared with RNH plastids. When RH plastids were lysed and washed, the number of plastoglobuli associated with thylakoid membranes decreased significantly, yet the PQA levels remained high. Room temperature fluorescence induction indicated that (a) there is no change in the size of the PQA pool immediately available for photochemistry in RNH and RH thylakoids and (b) there is a pool of oxidized PQA present in RNH and RH thylakoids which is not available for photochemistry. The accumulated PQA in RH thylakoids may reflect an increased nonphotochemical function such as regulation of thylakoid protein phosphorylation or protection against photoinhibition.     

Lipids of Leaves and Seeds

The lipid components of the chlorophyll lipoproteins isolated from the leaves of Cayratia japonica, Vicia sativa, and Artemisia princeps were separated and identified by column, thin-layer, and paper chromatographies. The lipids were mainly composed of carotenoids, quinones including plastoquinone, sterols and their esters, di- and monoglycerides, free fatty acids, chlorophylls and their degradation products, glycolipids including plant sulfolipids, and phospholipids, in which the glycolipids were predominant. The fatty acid composition was characteristic depending on each separated lipid component. Comparison of the lipid distributions was made between whole leaf and chlorophyll lipoprotein, and also between chlorophyll lipoproteins from young leaves and from full-grown leaves.     

Function of plastoquinones B and C as electron acceptors in Photosystem II and fatty acid analysis of plastoquinone B

We have found that in petroleum-ether extracted tobacco thylakoids, plastoquinone A (PQ-A) and plastoquinone C (PQ-C) had similar efficiency in restoration of oxygen-evolving activity, while plastoquinone B (PQ-B), which is a fatty acid ester of PQ-C, was about 50% less effective. This indicates that apart from PQ-A, PQ-C and to a smaller extent PQ-B may function as electron acceptors of Photosystem II (PS II). The DCMU inhibition curves for PQ-C and PQ-B were biphasic and an initial slow decline was followed by a sharp decrease in oxygen evolution yield with a 50% inhibition (I50) at 0.25 M DCMU. In the case of PQ-A (I50 = 0.20 M DCMU), the activity decreased gradually without the sharp transition. The corresponding inhibition curve for unextracted thylakoids, where all the native prenylquinones are present, shows an intermediate shape between PQ-A and PQ-C but with a higher I50, equal to 0.32 M, suggesting that the contribution of PQ-C as an electron acceptor of Photosystem II might be significant in thylakoid membranes with natural prenyllipid composition. -Tocopherol quinone showed no activity in the restoration of oxygen evolution in extracted thylakoids, indicating that it cannot accept electrons from PS II. The fatty acid composition of PQ-B isolated from maple leaves showed a high degree of saturated fatty acids like myristic and palmitic acid, and its unique composition indicates that it is a natural component of the thylakoid membrane.     

The non-photochemical reduction of plastoquinone in leaves

Although it is generally assumed that the plastoquinone pool of thylakoid membranes in leaves of higher plants is rapidly oxidized upon darkening, this is often not the case. A multiflash kinetic fluorimeter was used to monitor the redox state of the plastoquinone pool in leaves. It was found that in many species of plants, particularly those using the NAD-malic enzyme C₄ system of photosynthesis, the pool actually became more reduced following a light to dark transition. In some Amaranthus species, plastoquinone remained reduced in the dark for several hours. Far red light, which preferentially drives Photosystem I turnover, could effectively oxidize the plastoquinone pool. Plastoquinone was re-reduced in the dark within a few seconds when far red illumination was removed. The underlying mechanism of the dark reduction of the plastoquinone pool is still uncertain but may involve chlororespiratory activity.Abbreviations apparent F_o observed fluorescence yield after dark adaptation - F_m maximum fluorescence when all Q_A is fully reduced - F_o minimum fluorescence yield when Q_A is fully oxidized and non-photochemical quenching is fully relaxed - F_s steady state fluorescence yield - PPFD photosynthetic photon flux density - PQ plastoquinone - Q_A primary quinone acceptor of the Photosystem II reaction center - Q_B secondary quinone acceptor to the Photosystem II reaction center - F F_m minus F_s     

The Development of Chloroplast Ultrastructure and Hill Reaction Activity During Leaf Ontogeny in Different Maize (Zea Mays L.) Genotypes

Changes in Hill reaction activity (HRA) and ultrastructure of mesophyll cell (MC) chloroplasts were studied during the ontogeny of third leaf of maize plants using polarographic oxygen evolution measurement, transmission electron microscopy, and stereology. The chloroplast ultrastructure was compared in young (actively growing), mature, and senescing leaves of two different inbreds and their reciprocal F1 hybrids. Statistically significant differences in both HRA and MC chloroplast ultrastructure were observed between different stages of leaf ontogeny. Growth of plastoglobuli was the most striking characteristic of chloroplast maturation and senescence. The chloroplasts in mature and senescing leaves had a more developed system of thylakoids compared to the young leaves. Higher HRA was usually connected with higher thylakoid volume density of MC chloroplasts.     

Ontogenic Changes in Epicuticular Wax and Chloroplast Integrity of a Cotton (Gossypium hirsutum L.) Leaf

The progressive decline in cotton leaf photosynthesis with season could be accounted for by gaining an insight into ontogenic changes in chloroplast integrity and epicuticular wax ultrastructure. Therefore, the sequence of ultrastructural changes in chloroplast and epicuticular wax morphology were probed in 10-, 20-, 40-, and 60-d-old cotton (Gossypium hirsutum L.) leaves using electron microscopy. Scanning electron microscopy illustrated that the epicuticular wax on the periclinal walls of the convex epidermal cells occurred as striations and persisted as such during the course of leaf aging. The degree of wax spread, however, increased as the leaf progressed towards senescence. Transmission electron microscopy revealed that a 20-d-old photosynthetically active leaf possessed healthy chloroplasts (6.8 m long and an area of 9.7 m²) with absolute membrane integrity depicted by large appressed grana stacks of thylakoids interconnected by non-appressed stroma lamellae. The thylakoid membrane network was oriented parallel to the long axis of the chloroplast and a few small plastoglobuli (1.85 m²) scattered in the stroma. Conversely, membrane integrity was lost with leaf age after 20 d as evidenced by disruption of the grana and stroma lamellae. Concurrent with the membrane damage, extensive occlusion of chloroplast by several large spherical plastoglobuli (5.68 m²) occurred, the rate of occlusion increased with leafage distending the chloroplast as evidenced by proliferation of its cross-sectional area (12.8 m²). Of particular interest was the finding that the plastoglobuli ensued through the chloroplast envelope into the cytoplasm. The progressive loss of chloroplast membrane integrity coupled with increased leaf waxiness may have limited photosynthetic activities of cotton leaves during senescence.     

The Development of Chloroplast Ultrastructure and Hill Reaction Activity During Leaf Ontogeny in Different Maize (Zea Mays L.) Genotypes

Changes in Hill reaction activity (HRA) and ultrastructure of mesophyll cell (MC) chloroplasts were studied during the ontogeny of third leaf of maize plants using polarographic oxygen evolution measurement, transmission electron microscopy, and stereology. The chloroplast ultrastructure was compared in young (actively growing), mature, and senescing leaves of two different inbreds and their reciprocal F1 hybrids. Statistically significant differences in both HRA and MC chloroplast ultrastructure were observed between different stages of leaf ontogeny. Growth of plastoglobuli was the most striking characteristic of chloroplast maturation and senescence. The chloroplasts in mature and senescing leaves had a more developed system of thylakoids compared to the young leaves. Higher HRA was usually connected with higher thylakoid volume density of MC chloroplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Accumulation of pathogenesis-related proteins in barley leaf intercellular spaces during leaf senescence

Accumulation of the pathogenesis-related (PR) proteins localised in intercellular spaces of barley primary leaves, chlorophyll content, structure of chloroplasts, and photosynthesis were examined during natural and in vitro induced leaf senescence (cultivation of whole plants in the dark or detached leaves under nutrient deficiency). Some of PR proteins accumulated during natural senescence, but their accumulation pattern was different from those of pathogen-induced as well as during in vitro-induced senescence, which indicate different molecular bases of these processes. Photosynthetic rate and chlorophyll content indicate that natural senescence of barley primary leaves began from 15th day after sowing. In 35-d-old first leaves, the chloroplasts showed typical characteristics of senescence as significant decrease of size, greater grana, and prominent plastoglobuli. The chloroplasts contained more grana under in vitro induced senescence and they had reduced length in the dark. Correspondingly, accumulation of PR proteins was detectable on about the 15th day but the content of some PR proteins increased in later stages of senescence. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regreening of senescent Nicotiana leaves. II. Redifferentiation of plastids

Single senescent leaves attached to decapitated shoots of Nicotiana rustica L. regreened, especially when treated with cytokinin. Regreening caused an increase in leaf thickness, due to cell expansion. Senescent leaf plastids (gerontoplasts) were smaller than green chloroplasts, with degenerated membrane systems and stroma, and larger plastoglobuli. At advanced senescence, micrographs showed disintegrating gerontoplasts, reduced numbers of plastids were counted, and regreening became variable. The redevelopment of grana and stroma in regreening plastids was accelerated by cytokinin. All plastids in regreening leaves were identifiable as redifferentiating gerontoplasts because of their content of plastoglobuli and starch. Immunogold labelling showed significant association of POR with etioplasts in cotyledons, but with mature plastids in regreening leaves. No proplastids or dividing chloroplasts were observed in regreening leaves. Plastids numbers declined during senescence and did not increase again during regreening. It is concluded that the chloroplasts of regreening leaves arose by redifferentiation of gerontoplasts.Keywords: Chloroplasts, cytokinin, Nicotiana, senescence, regreening.      

Synthetic quinones influencing herbicide binding and Photosystem II electron transport. The effects of triazine-resistance on quinone binding properties in thylakoid membranes

We have analyzed the binding of synthetic quinones and herbicides which inhibit electron transport at the acceptor side of Photosystem II (PS II) of the photosynthetic electron-transport chain in thylakoid membranes. These data show that quinones and PS II-directed herbicides compete for binding to a common binding environment within a PS II region which functions as the Q⁻ / PQ oxidoreductase. We observed that (1) synthetic quinones cause a parallel inhibition of electron transport and [¹⁴C]herbicide displacement, and (2) herbicide binding is affected both by the fully oxidized and fully reduced form of a quinone. Quinone function and inhibitor binding were also investigated in thylakoids isolated from triazine-resistant weed biotypes. We conclude the following. (1) The affinity of the secondary accepting quinone, B, is decreased in resistant thylakoids. (2) The observation that the equilibrium concentration of reduced Q after transferring one electron to the acceptor side of PS II is increased in resistant as compared to susceptible chloroplasts may be explained both by a decrease in the affinity of PQ for the herbicide / quinone binding environment, and by a decrease of the midpont redox potential of the B / B⁻ couple. (3) The binding environment regulating quinone and herbicide affinity may be divided roughly into two domains; we suggest that the domain regulating quinone head-group binding is little changed in resistant membranes, whereas the domain-regulating quinone side-group binding (and atrazine) is altered. This results in increased inhibitory activity of tetrachloro-p-benzoquinone and phenolic herbicides, which are hypothesized to utilize the quinone head-group domain. The two domains appear to be spatially overlapping because efficient atrazine displacement by tetrachloro-p-benzoquinone is observed.     

Oleosomes in flag leaves of wheat; their distribution,composition and fate during senesence and rust-infection

Oleosomes, up to 14m in diameter, were found in mesophyll and bundle sheath cells of the flag and lower leaves of wheat cv Professeur Marchal. They develop in flag leaves at least 10 d before anthesis, possibly from fatty acids secreted by the plastids, and persist in mature and senescing leaf tissue. Oleosomes are bordered with an osmiophilic layer rather than a unit membrane. The major lipids of oleosomes, isolated 20 d after anthesis, are triacylglycerols (50%) and sterol or wax exter (34%). The dominant fatty acids of both lipid classes are plamitic (16:0) and stearic (18:0) acids which accounts for the low osmiophilia of the oleosomes. The function of the oleosomes is unknown but they may act as short-term energy reserves. Oleosomes persist in leaves infected with brown rust, even in cells penetrated by haustoria. Yellowish-brown oleosomes found in senescing and rust-infected leaves may be formed by the release and coalescence of pigmented plastoglobuli.     

Light-induced quinone reduction in photosystem II

The photosystem II core complex is the water:plastoquinone oxidoreductase of oxygenic photosynthesis situated in the thylakoid membrane of cyanobacteria, algae and plants. It catalyzes the light-induced transfer of electrons from water to plastoquinone accompanied by the net transport of protons from the cytoplasm (stroma) to the lumen, the production of molecular oxygen and the release of plastoquinol into the membrane phase. In this review, we outline our present knowledge about the "acceptor side" of the photosystem II core complex covering the reaction center with focus on the primary (Q(A)) and secondary (Q(B)) quinones situated around the non-heme iron with bound (bi)carbonate and a comparison with the reaction center of purple bacteria. Related topics addressed are quinone diffusion channels for plastoquinone/plastoquinol exchange, the newly discovered third quinone Q(C), the relevance of lipids, the interactions of quinones with the still enigmatic cytochrome b559 and the role of Q(A) in photoinhibition and photoprotection mechanisms. This article is part of a Special Issue entitled: Photosystem II.     

Carotenoid metabolism during chloroplast to chromoplast transformation in Capsicum annuum fruit

Lipid and carotenoid metabolism associated with the structural transformations of the plastids during the ripening of Capsicum annuum fruits were studied. The early ripening stage is characterized by chloroplasts with a typical grana-intergranal structure and a highly developed peripheral reticulum. At a later stage the thylakoid system is disintegrated and replaced by non-chlorophyllous single thylakoids, derived in part from the inner envelope membrane. First, these changes coincide with a loss of galactolipids, a slow increase in phospholipid content, a decrease in the ratio galactolipids/phospholipids and in the galactosyl transferase activities on a protein basis. Second, these changes correlate with an enhanced accumulation of keto-carotenoids. When the phospholipid environment is disturbed by difluorodinitrobenzene or phospholipases, the biosynthesis of phytoene, -carotene, and capsanthin is inhibited; phospholipase D has a greater effect. The role of a phospholipid environment in carotenoid biosynthesis is discussed.Abbreviations DFDNB 1.5-difluoro-2.4-dinitrobenzene - BSA bovine serum albumine - DTT dithiothreitol     

Cytokinin promotes catalase and ascorbate peroxidase activities and preserves the chloroplast integrity during dark-senescence

Increased oxidative stress displayed during dark-senescence of wheat leaves (Triticum aestivum L.) is caused not only by the increased levels of radicals but also by a loss of antioxidant capacity. Mature leaves were incubated in 6-benzylaminopurine (BAP 10⁻⁴ M) or water (control) during 6 d in the dark. The senescence-delaying effect of BAP was associated with the retention of the chloroplast structure, 60% of the initial content of chlorophyll (Chl) and 77% of the initial content of protein. BAP reduced the degradation of the light-harvesting chlorophyll a/b binding protein (LHCP-2), and the large (LSU) and small subunits (SSU) of Rubisco. Our results indicated that the presence of the NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1) was not promoted by the cytokinin, leading to the conclusion that BAP maintains the level of Chl, preventing its degradation, rather than inducing Chl biosynthesis. The internal structure of chloroplasts was maintained in BAP-treated leaves for up to 6 d, with well-organized grana thylakoids and small plastoglobuli; in contrast, chloroplasts of control leaves deteriorated rapidly from day 4 with disorganized internal membranes, and more and larger plastoglobuli. BAP increased the activities of catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) and reduced the level of H₂O₂ in the delayed-senescence tissue. The present research indicates that BAP reduces levels of reactive oxygen species (ROS), and enhances the activity of antioxidant enzymes (CAT, APX). Our results suggest that BAP protects the cell membranes and the photosynthetic machinery from oxidative damage during delay of senescence in the dark.     

Temperature-dependent changes in Photosystem II heterogeneity support a cycle of Photosystem II during photoinhibition

High light treatments were given to attached leaves of pumpkin (Cucurbita pepo L.) at room temperature and at 1°C where the diffusion- and enzyme-dependent repair processes of Photosystem II are at a minimum. After treatments, electron transfer activities and fluorescence induction were measured from thylakoids isolated from the treated leaves. When the photoinhibition treatment was given at 1°C, the Photosystem II electron transfer assays that were designed to require electron transfer to the plastoquinone pool showed greater inhibition than electron transfer from H₂O to paraphenyl-benzoquinone, which measures all PS II centers. When the light treatment was given at room temperature, electron transfer from H₂O to paraphenyl-benzoquinone was inhibited more than whole-chain electron transfer. Variable fluorescence measured in the presence of ferricyanide decreased only during room-temperature treatments. These results suggest that reaction centers of one pool of Photosystem II, non-Q_B-PS II, replace photoinhibited reaction centers at room temperature, while no replacement occurs at 1°C. A simulation of photoinhibition at 1°C supports this conclusion.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1,-dimethylurea - DCPIP dichlorophenol-indophenol (2,6-dichloro-4((4-hydroxyphenyl)imino)-2,5-cyclohexadien-1-one) - DPC diphenyl carbazide (2,2-diphenylcarbonic dihydrazide) - FeCN ferricyanide (hexacyanoferrate(III)) - _app apparent quantum yield of photosynthetic oxygen evolution - MV methyl viologen (1,1-dimethyl-4,4-bipyridinium dichloride) - PPBQ phenyl-p-benzoquinone - PPFD photosynthetic photon flux density - PQ pool plastoquinone - Q_B secondary quinone acceptor of PS II - RT room temperature - WC whole chain electron transfer