Ultrastructure and Dimensions of Chloroplasts in Leaves of Three Maize (Zea mays L.) Inbred Lines and their F1 Hybrids Grown Under Moderate Chilling Stress

Influence of moderate chilling stress on vascular bundle sheath cell (BSC) and especially mesophyll cell (MC) chloroplasts of mature maize leaves was studied by electron microscopy and stereology. Plants of two inbred lines of maize, differing in their photosynthetic activity, and their F₁ hybrids were cultivated during autumn in heated or unheated glasshouse. Generally, chilling temperatures resulted mainly in the decrease in stereological volume density (VD) of both granal and intergranal thylakoids of MC chloroplasts, while the ratio of granal to all thylakoids (granality) was less affected. The VD of peripheral reticulum and plastoglobuli usually increased after cold treatment of plants. The volume of MC chloroplasts usually increased under chilling stress, the shape of the chloroplasts changed only slightly. The ultra-structure of chloroplasts differed between individual genotypes; chilling-stressed hybrid plants showed positive heterosis particularly in the granal thylakoids' VD of MC chloroplasts.     

Light Modulation and Localization of Sucrose Phosphate Synthase Activity between Mesophyll Cells and Bundle Sheath Cells in C(4) Species

Experiments were conducted with several Panicum species (representing the different C₄ subtypes) to examine the light modulation of sucrose phosphate synthase (SPS) activity and the effect of illumination on the distribution of SPS activity between mesophyll cells (MC) and bundle sheath cells (BSC). Activity of SPS in the light decreased in the order: C₄ > C₃-C₄ intermediate > C₃. In illuminated leaves, SPS activities were similar among the three C₄ subtypes, but SPS activity was higher for NAD-malic enzyme (NAD-ME) species with centripetal chloroplasts in BSC (NAD-ME(P) species) than for NAD-ME species with centrifugal chloroplasts in BSC (NAD-ME(F) species). Transfer of plants into darkness for 30 minutes resulted in decreased SPS activity for all species tested except Panicum bisulcatum (C₃ species) and Panicum virgatum (NAD-ME(P) species) which showed little or no change. All C₄ subtypes had some SPS activity both in MC and BSC. In the light, SPS activity was mainly in the MC for NADP-ME, NAD-ME(F) and phosphoenolpyruvate carboxykinase species, while it was mainly in the BSC for NAD-ME(P) species. In the dark, for all C₄ subtypes, SPS activity in the MC was decreased to a greater extent than that in the BSC. It is intriguing that NAD-ME(F) and NAD-ME(P) species differed in the activity and distribution of SPS activity between MC and BSC, although they are otherwise identical in the photosynthetic carbon assimilation pathway. Diurnal changes in SPS activity in the MC and BSC were also examined in maize leaves. SPS activity in the MC in maize leaves was high and relatively constant throughout the middle of the light period, dropped rapidly after sunset and increased again prior to the light period. On the other hand, SPS activity in the BSC was lower and changed more coincidently with light intensity than that in the MC. The results suggested that light activation of SPS activity located in the BSC may require higher irradiance for saturation than the SPS in the MC. We conclude that SPS may function in both MC and BSC for sucrose synthesis in the light, particularly at high light intensity, while in the dark, the major function may be in the BSC during starch degradation.     

Distribution and Properties of Glycolate Oxidase from Bundle Sheath and Mesophyll Cells of Green Amaranth Leaves (<Emphasis Type="Italic">Amaranthus retroflecsus</Emphasis>)

Separation of mesophyll and bundle sheath cells (MC and BSC) from the leaves of green amaranth (Amaranthus retroflexus L.) showed that glycolate oxidase (GO, EC 1.1.3.35) is located predominately in BSC (on the average, 84.5% of the total activity). Three peaks of GO activity were detected following the elution from a DEAE-fractogel column. The first peak corresponded to the isoform located in BSC, the second peak had dual location, and the third one was associated with MC fraction. Elaborated flow sheet of GO purification from the amaranth leaves produced highly purified (by 63.5 times) isoforms from MC and BSC with specific activity of 0.54 EU/mg protein. It was also shown that GO from MC has greater affinity for glycolate, with the K _M values for GO from BSC and MC being 58 and 20 µM, respectively. Intermediates of the Krebs cycle were shown to affect the GO activity from MC and BSC: succinate suppressed and isocitrate activated GO.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 622–627.Original Russian Text Copyright © 2005 by Eprintsev, Ivent’ev, Popov.     

Comparative analysis of thylakoid protein complexes in the mesophyll and bundle sheath cells from C3, C4 and C3–C4 Paniceae grasses

To better understand the coordination between dark and light reactions during the transition from C₃ to C₄ photosynthesis, we optimized a method for separating thylakoids from mesophyll (MC) and bundle sheath cells (BSCs) across different plant species. We grew six Paniceae grasses including representatives from the C₃, C₃–C₄ and C₄ photosynthetic types and all three C₄ biochemical subtypes [nicotinamide adenine dinucleotide phosphate‐dependent malic enzyme (NADP‐ME), nicotinamide adenine dinucleotide‐dependent malic enzyme (NAD‐ME) and phosphoenolpyruvate carboxykinase (PEPCK)] in addition to Zea mays under control conditions (1000 μmol quanta m^?2 s^?1 and 400 ppm of CO₂). Proteomics analysis of thylakoids under native conditions, using blue native polyacrylamide gel electrophoresis followed by liquid chromatography‐mass spectrometry (LC‐MS), demonstrated the presence of subunits of all light‐reaction‐related complexes in all species and cell types. C₄ NADP‐ME species showed a higher photosystems I/II ratio and a clear accumulation of the NADH dehydrogenase‐like complexes in BSCs, while Cytb₆f was more abundant in BSCs of C₄ NAD‐ME species. The C₄ PEPCK species showed no clear differences between cell types. Our study presents, for the first time, a good separation between BSC and MC for a C₃–C₄ intermediate grass which did not show noticeable differences in the distribution of the thylakoid complexes. For the NADP‐ME species Panicum antidotale, growth at glacial CO₂ (180 ppm of CO₂) had no effect on the distribution of the light‐reaction complexes, while growth at low light (200 μmol quanta m^?2 s^?1) promoted the accumulation of light‐harvesting proteins in both cell types. These results add to our understanding of thylakoid distribution across photosynthetic types and subtypes, and introduce thylakoid distribution between the MC and BSC of a C₃–C₄ intermediate species.     

Aggregative movement of C4 mesophyll chloroplasts is promoted by low CO2 under high intensity blue light

• C₄ plants supply concentrated CO₂ to bundle sheath (BS) cells, improving photosynthetic efficiency by suppressing photorespiration. Mesophyll chloroplasts in C₄ plants are redistributed toward the sides of the BS cells (aggregative movement) in response to environmental stresses under light. Although this chloroplast movement is common in C₄ plants, the significance and mechanisms underlying the aggregative movement remain unknown.
• Under environmental stresses, such as drought and salt, CO₂ uptake from the atmosphere is suppressed by closing stomata to prevent water loss. We hypothesized that CO₂ limitation may induce the chloroplast aggregative movement. In this study, the mesophyll chloroplast arrangement in a leaf of finger millet, an NAD-malic enzyme type C₄ plant, was examined under different CO₂ concentrations and light conditions.
• CO₂ limitation around the leaves promoted the aggregative movement, but the aggregative movement was not suppressed, even at the higher CO₂ concentration than in the atmosphere, under high intensity blue light. In addition, mesophyll chloroplasts did not change their arrangement under darkness or red light.
• From these results, it can be concluded that CO₂ limitation is not a direct inducer of the aggregative movement but would be a promoting factor of the movement under high intensity blue light.

     

The Ultrastructure of Chloroplasts,Content of Photosynthetic Pigments,and Photochemical Activity of Maize (Zea mays L.) as Influenced by Different Concentrations of the Herbicide Amitrole

The effect of three different concentrations of amitrole (AM), a bleaching herbicide affecting carotenogenesis, on chloroplast ultrastructure, photosynthetic pigment contents, and photochemical activity was studied in two maize genotypes differing in photosynthetic characteristics. The content of photosynthetic pigments in leaves of plants treated with low (20 M) AM concentration was similar to control plants and no damaging effect of the herbicide on the ultrastructure of either mesophyll (MC) or bundle-sheath (BSC) cell chloroplasts was observed. Higher (60 and 120 M) concentrations of AM caused a significant decrease in the content of carotenoids (especially xanthophylls), which was followed by photooxidative destruction of chlorophylls and some alterations of chloroplast ultrastructure. MC chloroplasts appeared more sensitive to the damaging effect of AM compared to BSC chloroplasts. A significant decrease in the amount of both granal and intergranal thylakoids in MC chloroplasts was observed with the increasing concentration of AM. As regards BSC chloroplasts, rapid decrease in the volume density of starch inclusions was found in plants treated with higher concentrations of AM. When 120 M AM was used, both MC and BSC chloroplasts contained just a few thylakoid membranes that were strongly altered. The changes in the ultrastructure of MC chloroplasts were accompanied by the changes in their photochemical activity. The formation of chloroplast protrusions after treatment of plants with AM as well as in control plants was also observed.     

The effect of nitrogen and phosphorus on starch accumulation and net photosynthesis in two variants of Panicum maximum Jacq.

Abstract. Two anatomical variants of Panicum maximum Jacq. were observed to accumulate an unusually large number of starch grains in the bundle sheath chloroplasts when grown under controlled environmental conditions in a nutrient medium containing a low level of nitrate nitrogen (20 mg N dm⁻³ as KNO₃). When these plants were placed under dark conditions the chloroplasts were destarched, but exhibited a marked distortion of the thylakoid membranes. Under a higher level of nitrate nitrogen supply (200 mg N dm⁻³ as KNO₃) the number of starch grains was markedly reduced compared to that observed above in both plant variants. When the nitrogen was supplied as ammonium nitrogen (200 mg N dm⁻³ as NH₄Cl) there was again a high level of starch in the bundle sheath chloroplasts, the level being only slightly lower than that observed at the low KNO₃ supply. An unusually large number of starch grains accumulated in the bundle sheath chloroplasts in the absence of added phosphorus in the nutrient medium, in the presence of the higher nitrate nitrogen level. It is suggested that the increased starch accumulation results from a reduced trans-location of Calvin cycle intermediates out of the chloroplasts into the cytoplasm and that both nitrate nitrogen and phosphorus may play an important role in this process. A good correlation between high net photosynthetic activity and low bundle sheath starch content was observed. Nutrient medium requirements favouring low starch content in chloroplasts also favoured high net photosynthetic rates.     

Electron flow accompanying the ascorbate peroxidase cycle in maize mesophyll chloroplasts and its cooperation with linear electron flow to NADP+ and cyclic electron flow in thylakoid membrane energization

Potential roles for cyclic and pseudocyclic electron flow in C₄ plants are to provide ATP for the C₄ cycle and, under excess light, to down-regulate PS II activity through membrane energization. Intact mesophyll chloroplasts of maize were used to evaluate forms of electron transport including the Mehler peroxidase reaction (linear electron flow to O₂, formation of H₂O₂ which is reduced by ascorbate, and linear flow linked to reduction of oxidized ascorbate). Addition of H₂O₂ to isolated chloroplasts in the light in the presence of an uncoupler induced Photosystem (PS) II activity, as determined from increases in photochemical quenching of chlorophyll fluorescence (q_p) and the quantum yield of PS II. H₂O₂ also induced dissipation of energy by thylakoid membrane energization and non-photochemical fluorescence quenching (q_n), which was inhibited by addition of an uncoupler. These effects of H₂O₂ on q_p and q_n were inhibited by addition of KCN, an inhibitor of ascorbate peroxidase. The results suggest that H₂O₂ is reduced via ascorbate, and that the oxidized ascorbate is then reduced by linear electron flow contributing to photochemistry and thylakoid membrane energization. Evidence for function of pseudocyclic electron flow via the Mehler peroxidase reaction was obtained with only oxygen as an electron acceptor, as well as in the presence of oxaloacetate a natural electron acceptor in C₄ photosynthesis. KCN decreased q_p and PS II yield in the absence and presence of oxaloacetate and, in the former case, it severely reduced q_{_n}. KCN also decreased pH formation across the thylakoid membrane based on its decrease in the light-induced quenching of 9-aminoacridine fluorescence, particularly in the absence of oxaloacetate. Antimycin A, an inhibitor of cyclic electron flow, also diminished pH formation. These results provide evidence for shared energization of thylakoid membranes by the Mehler peroxidase reaction, cyclic electron flow, and linear electron flow linked to the C₄ pathway.     

CO2 uptake and transport in leaf mesophyll cells

Abstract The acquisition of inorganic carbon for photosynthetic assimilation by leaf mesophyll cells and chloroplasts is discussed with particular reference to membrane permeation of CO₂ and HCO^?₃. Experimental evidence indicates that at the apoplast pH normally experienced by leaf mesophyll cells (pH 6–7) CO₂ is the principal species of inorganic carbon taken up. Uptake of HCO^?₃ may also occur under certain circumstances (i.e. pH 8.5), but its contribution to the net flux of inorganic carbon is small and HCO^?₃ uptake does not function as a CO₂-concentrating mechanism. Similarly, CO₂ rather than HCO^?₃ appears to be the species of inorganic carbon which permeates the chloroplast envelope. In contrast to many C₃ aquatic plants and C₄ plants, C₃ terrestrial plants lack specialized mechanisms for the acquisition and transport of inorganic carbon from the intercellular environment to the site of photosynthetic carboxylation, but rely upon the diffusive uptake of CO₂.     

盐胁迫对玉米叶片叶肉细胞生物膜超微结构的影响

研究了NaCl胁迫对玉米叶肉细胞生物膜超微结构的影响. 结果表明：NaCl胁迫破坏了玉米叶片叶肉细胞生物膜的正常结构,50 mmol·L^-1 NaCl处理胁迫下,玉米叶肉细胞核膜,线粒体膜,细胞膜,叶绿体膜,液泡膜都受到不同程度的破坏,叶绿体基粒类囊体膨胀,间质片层空间增大,片层紊乱。100 mmol·L^-1 NaCl处理胁迫下,质膜,液泡膜,线粒体,叶绿体都受到严重的破坏。细胞质膜破坏,破损的叶绿体充斥在细胞间隙中;叶绿体外膜破坏,甚至解体消失,叶肉细胞中充满膜结构,基粒排列方向改变,垛叠层数减少,基粒和基质片层界限模糊不清,有的基粒解体消失,甚至叶绿体完全解体;核膜破坏、解体,核中的染色质高度凝缩;线粒体的数量增多,线粒体膜破坏,脊的数量减少,甚至整个线粒体破损解体;液泡膜破坏;由于各种生物膜的破坏,使细胞内充满许多囊状小泡、多泡体或斑层小体;叶肉细胞发生严重的质壁分离,严重时发生细胞壁断裂;甚至整个细胞溶解。     

Leaf anatomy and C4 photosynthetic enzymes in three reed ecotypes

Differences in leaf interveinal distances, chloroplasts distribution in bundle sheath cells (BSC) and activities of C₄ photosynthetic enzymes in the leaves of three ecotypes of Phragmites communis Trinius, namely swamp reed (SR), heavy salt meadow reed (HSMR) and dune reed (DR), occurring in the desert region of northwest China were investigated. The two terrestrial ecotypes, DR and HSMR, had denser vascular system, more and longer BSC chloroplasts and higher capacity of CO₂ concentrating mechanism of NAD-ME subtype as compared with the SR ecotype. The enhanced NADP-ME pathway in the HSMR might contribute to its adaptation to the salinity habitat.     

Glutamine synthetase from mesophyll and bundle sheath maize cells: isoenzyme complements and different sensitivities to phosphinothricin

 Anion-exchange FPLC has been used to resolve the isoforms of glutamine synthetase (GS, EC 6.3.1.2) from Zea mays mesophyll (MC) and bundle sheath cells (BSC). Two different isoforms were detected in both types of photosynthetic cells. The predominantly active isoform was GS1 (61%) in MC and GS2 (67%) in BSC. The relative contribution of GS1 and GS2 to the overall GS activity in BSC in maize here reported resembles the proportion described for most C3 plants. Differences among these isoforms in terms of their susceptibility to phosphinothricin (PPT), an analogue of glutamate and known inhibitor of GS, were found. The GS1 isoenzyme from MC was the most sensitive form, being inhibited by 50% at approximately 2.0 μM DL-PPT, whereas the GS2 from BSC presented the highest tolerance to the inhibitor (I₅₀=30 μM). The transferase-to-semibiosynthetic activity ratio for the MC isoforms, which was higher than the ratio for the BSC isoforms, and the differences shown by the isoforms in susceptibility to PPT predict important differences in the biochemical properties and regulation of GS isoenzymes. In this regard, the cytoplasmic isoenzymes, and especially the one in MC, due to its relatively high contribution to mesophyll cell GS activity, could play a vital role in nitrogen metabolism in maize. Received: 1 December 1999 / Revised: 7 February 2000 / Accepted: 23 February 2000     

Chloroplast ultrastructure and thylakoid polypeptide composition are affected by different salt concentrations in the halophytic plant Arthrocnemum macrostachyum

The effect of different external salt concentrations, from 0 mM to 1030 mM NaCl, on photosynthetic complexes and chloroplast ultrastructure in the halophyte Arthrocnemum macrostachyum was studied. Photosystem II, but not Photosystem I or cytochrome b6/f, was affected by salt treatment. We found that the PsbQ protein was never expressed, whereas the amounts of PsbP and PsbO were influenced by salt in a complex way. Analyses of Photosystem II intrinsic proteins showed an uneven degradation of subunits with a loss of about 50% of centres in the 0 mM NaCl treated sample. Also the shape of chloroplasts, as well as the organization of thylakoid membranes were affected by NaCl concentration, with many grana containing few thylakoids at 1030 mM NaCl and thicker grana and numerous swollen thylakoids at 0 mM NaCl. The PsbQ protein was found to be depleted also in thylakoids from other halophytes.     

Development of the Kranz structure during leaf growth in C4 Euphorbia maculate

The development of the Kranz structure was investigated in leaves of C₄ Euphorbia maculata using electron microscopy. Four leaf stages, i.e., primordial, immature, young, and mature, were examined, based on the photosynthetic tissue that surrounded the veins. The examination revealed how cells differentiated into distinct bundle sheath cells (BSCs) and mesophyll cells (MCs). Specialization of the BSCs was invariably associated with the development of the veins as well as the MCs. Precursors for BSC and MC were recognizable fairly early, at the immature stage, according to their position and differential enlargement Once these precursors were delimited from the procambial area, differentiation into each cell type occurred synchronously, in a coordinated manner. All cells enlarged as they were displaced from the Kranz precursor area, but the BSC precursors were initially larger and remained relatively larger than the other cell types throughout leaf development The developmental changes sharply distinguished BSCs from the adjacent MCs at the onset of Kranz formation and continued until maturity. Chloroplast enlargement also occurred during cell displacement, but the rate of enlargement was greater in BSCs, resulting in larger chloroplasts at later stages. However, no significant structural differences were detected among the chloroplasts of BSC and MC in the early stages. Most of the specialized features appeared at the young-leaf stage; structural dimorphism became prominent at the later stages. This enhanced development of the BSC chloroplasts was correlated with asymmetric distribution of cellular components. In addition, the BSC formed thin primary pit fields with numerous plasmodesmata. Peripheral reticulum was present, but generally was not conspicuous. We also discuss the characteristics of leaf anatomy and ultrastructure inE. maculata as they relate to the C₄ photosynthetic pathway.     

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pH_in established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pH_out = 7.8, we found that pH_in ≈ 5.4-5.7 in the state of photosynthetic control, and pH_in ≈ 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8-2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H⁺/ATP is n ≥ 4-4.7.     

Plants under Climatic Stress: II. Low Temperature, High Light Effects on Chloroplast Ultrastructure

Mesophyll chloroplasts of the C₄-pathway grasses Sorghum and Paspalum and of the C₃-pathway legume soybean undergo ultrastructural changes under moderate light intensities (170 w·m⁻², 400-700 nanometers) at a tme when photosynthesis is much reduced by low temperature (10 C). The pattern of ultrastructural change was similar in these species, despite some differences in the initial sites of low temperature action on photosynthesis and differences in their mechanisms of CO₂ fixation. Starch grains in the chloroplasts rapidly reduce in size when chilling stress is applied. At or before the time starch grains completely disappear the membranes of the individual stromal thylakoids close together, reducing the intraspace between them while the chloroplast as a whole begins to swell. Extensive granal stacking appears to hold the thylakoids in position for some time, causing initial swelling to occur in the zone of the peripheral reticulum, when present. At more advanced stages of swelling the thylakoid system unravels while the thylakoid intraspaces dilate markedly. Initial thylakoid intraspace contraction is tentatively ascribed to an increase in the transmembrane hydrogen ion gradient causing movement of cations and undissociated organic acids from the thylakoid intraspace to the stroma. Chloroplast swelling may be caused by a hold-up of some osmotically active photosynthetic product in the chloroplast stroma. After granal unraveling and redilation of the thylakoid intraspaces, chloroplasts appear similar to those isolated in low salt hypotonic media. At the initial stages of stress-induced ultrastructural change, a marked gradient in degree of chloroplast swelling is seen within and between cells, being most pronounced near the surface of the leaf directly exposed to light.     

盐胁迫下苜蓿中盐蛋白的诱导产生

盐胁迫下苜蓿叶片中蛋白质的合成受到抑制,而其离体叶绿体中蛋白质合成增强,ABA阻碍了后者的蛋白质合成。NaCl胁迫下,“松江”和“肇东”两品种的根和叶中均无新多肽出现。在盐敏感的“松江”品种离体叶绿体中,NaGl诱导70,65,60和43kD4种多肽产生,ABA诱导60和17kD两种多肽产生;在较抗盐的“肇东”品种离体叶绿体中,NaGl诱导83,80kD和43kD3种多肽产生,但100mmol/L NaCl并不诱导83kD多肽出现,ABA无明显作用。两品种的43kD多肽和肇东品种的80kD多肽都存在于类囊体膜上,而松江品种的60kD多肽则存在于叶绿体间质中。     

Assessing photosystem I and II distribution in leaves from C4 plants using confocal laser scanning microscopy

Images of chlorophyll fluorescence emitted at wavelengths above and below 700 nm were recorded from leaf sections of C₄ species using confocal laser scanning microscopy (LSM). We investigated species exhibiting both NAD-malic enzyme (NAD-ME) C₄ photosynthesis and NADP-malic enzyme (NADP-ME) C₄ photosynthesis. Comparing LSM fluorescence of leaf sections with flow-cytometrically determined fluorescence from individual chloroplasts revealed that LSM fluorescence was distorted by the optical properties of leaf sections. Leaf section fluorescence, when corrected by transmission data derived from light transmission images, agreed with flow cytometry data. The corrected LSM fluorescence yielded information on the distribution of the individual photosystems in the C₄ leaf sections: PSII concentrations in bundle sheath cells were elevated in NAD-ME species but diminished in most of the NADP-ME species investigated. The NADP-ME species, Arundinella hirta, however, showed normal PSII and increased PSI concentration in bundle sheath chloroplasts. Finally, a gradient of PSI was observed within the bundle sheath cells from Euphorbia maculata.