Flow cytometry of mesophyll and bundle sheath chloroplast thylakoids of maize (Zea mays L.)

Pfündel, Erhard; Meister, Armin

doi:10.1002/(sici)1097-0320(19960201)23:2<97::aid-cyto2>3.0.co;2-i

Cited by 16 publications

(7 citation statements)

References 29 publications

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“…For fluorescence measurements, diluted leaf powder was prepared according to Weis (1985) with modifications described by Pfündel & Pfeffer (1997). Fluorescence excitation spectra were recorded at a low temperature (77 K) using an LS 50B (Perkin Elmer, Buckinghamshire, UK) as specified earlier ( Pfündel & Meister 1996).…”

Section: Methodsmentioning

confidence: 99%

Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants

et al. 2000

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SummaryThe chelation of Fe 2+ and Mg 2+ ions forms protoheme IX and Mg-protoporphyrin IX, respectively, and the latter is an intermediate in chlorophyll synthesis. Active magnesium protoporphyrin IX chelatase (Mg-chelatase) is an enzyme complex consisting of three different subunits. To investigate the function of the CHL I subunit of Mg-chelatase and the effects of modi®ed Mg-chelatase activity on the tetrapyrrole biosynthetic pathway, we characterized N. tabacum transformants carrying gene constructs with the Chl I cDNA sequence in antisense and sense orientation under the control of the CaMV 35S promoter. Both elevated and diminished levels of Chl I mRNA and Chl I protein led to reduced Mgchelatase activities, re¯ecting a perturbation of the assembly of the enzyme complex. The transformed plants did not accumulate the substrate of Mg-chelatase, protoporphyrin IX, but the leaves contained less chlorophyll and possessed increased chlorophyll a/b ratios, as well as a de®ciency of light-harvesting chlorophyll binding proteins of photosystems I and II. The expression and activity of several tetrapyrrolic enzymes were reduced in parallel to lower the Mg-chelatase activity. Consistent with the lower chlorophyll contents, the rate-limiting synthesis of 5-aminolevulinate was also decreased in the transgenic lines analyzed. The consequence of reduced Mg-chelatase on early and late steps of chlorophyll synthesis, and on the organization of light harvesting complexes is discussed.

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Section: Methodsmentioning

confidence: 99%

Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants

et al. 2000

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“…Cross‐sections of 20–40 μm thickness were prepared using a cryo‐microtome (Leica CM3050‐Cryostat, Leica Instruments GmbH) at –15 °C, and collected on poly‐ L ‐lysine‐coated slides (Sigma, St Louis, Missouri, USA). Probably, the freeze–thaw cycle could affect the chloroplast ultra‐structure; however, flow cytometric investigations showed that even membrane particles released from chloroplasts were unaltered in their fluorescence properties ( Pfündel & Meister 1996). Hence, structural changes during sample preparation do not affect the fluorescence signal that we consider here.…”

Section: Methodsmentioning

confidence: 99%

“…Preparations enriched in either mesophyll or bundle sheath chloroplasts were obtained according to Höfer, Santore & Westhoff (1992) with the modifications specified in Pfündel & Meister (1996). Flow cytometrical analysis of chloroplasts was done with a FACStar Plus sorting flow cytometer (Becton Dickinson, San Jose, California, USA) as described previously ( Pfündel & Meister 1996). Chlorophyll fluorescence of single particles was excited by 488 nm light from an argon ion laser operated at 500 mW output power.…”

Section: Methodsmentioning

confidence: 99%

“…Hence, a coarse estimate on the correctness of the fluorescence from leaf sections can be made by comparing the measured ratios of long‐wavelength to short‐wavelength chlorophyll fluorescence with the known PSI/PSII ratios. Secondly, for the plants chosen, the fluorescence characteristics of the individual chloroplasts from the two photosynthetic domains are known from earlier investigations using flow cytometry ( Pfündel & Meister 1996; Pfündel, Nagel & Meister 1996). Therefore, we are able to carry out a detailed analysis of fluorescence re‐absorption by comparing the fluorescence from leaf sections with that of single chloroplasts.…”

Section: Introductionmentioning

confidence: 99%

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

Pfündel

Neubohn

1999

Plant Cell & Environment

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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.

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“…Although grossly disorganized, there was less evidence of chloroplast swelling in the BS cells of lls1 leaves. This may be because of the differential sensitivities of the chloroplast types to ROS or a differential ability of the BS chloroplasts to produce ROS because they lack the oxygen-generating PSII found in mesophyll chloroplasts (Pfuendel and Meister, 1996;Kingston-Smith and Foyer, 2000). Lower ROS production could result in less damage to BS cells and this could explain why the autocatalytic spread of lls1 lesions is retarded near vascular elements.…”

Section: Loss Of Chloroplast Structural Integrity Is a Key Event In Lmentioning

confidence: 99%

Light-Dependent Death of Maize lls1 Cells Is Mediated by Mature Chloroplasts

et al. 2002

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We reported previously the isolation of a novel cell death-suppressing gene from maize (Zea mays) encoded by the Lls1 (Lethal leaf spot-1) gene. Although the exact metabolic function of LLS1 remains elusive, here we provide insight into mechanisms that underlie the initiation and propagation of cell death associated with lls1 lesions. Our data indicate that lls1 lesions are triggered in response to a cell-damaging event caused by any biotic or abiotic agent or intrinsic metabolic imbalance-as long as the leaf tissue is developmentally competent to develop lls1 lesions. Continued expansion of these lesions, however, depends on the availability of light, with fluence rate being more important than spectral quality. Double-mutant analysis of lls1 with two maize mutants oil-yellow and iojap, both compromised photosynthetically and unable to accumulate normal levels of chlorophyll, indicated that it was the light harvested by the plant that energized lls1 lesion development. Chloroplasts appear to be the key mediators of lls1 cell death; their swelling and distortion occurs before any other changes normally associated with dying cells. In agreement with these results are indications that LLS1 is a chloroplast-localized protein whose transcript was detected only in green tissues. The propagative nature of light-dependent lls1 lesions predicts that cell death associated with these lesions is caused by a mobile agent such as reactive oxidative species. LLS1 may act to prevent reactive oxidative species formation or serve to remove a cell death mediator so as to maintain chloroplast integrity and cell survival.lls1 (lethal leaf spot-1) is a maize (Zea mays) mutation, characterized by the formation of necrotic spots that expand continuously to kill the entire leaf and eventually the whole plant. The developmentally programmed phenotype of lls1 manifests in a cell autonomous fashion as evidenced by the discrete border between mutant and revertant tissue in sectored plants (Gray et al., 1997) and is suggestive of the involvement of an endogenous program in lls1 cell death. Because this mutation is inherited in a strictly recessive fashion, it is likely that the wildtype Lls1 gene functions to positively maintain cell homeostasis (Ullstrup and Troyer, 1967; Johal et al., 1994). The Lls1 gene has been cloned. Although it appears to encode a novel protein specific to plants, it does have two motifs, a Rieske-type Fe-sulfur center and a mononuclear non-heme Fe-binding site, that are found in the aromatic ring-hydroxylating dioxygenases of bacteria. Because of the fact that the biochemical function of these enzymes is to degrade aromatic hydrocarbons, we hypothesized previously that LLS1 may also work by breaking down a phenolic mediator of cell death in plants (Gray et al., 1997). This proposal remains contentious, however, because the nature of the substrate, if any, for LLS1 remains unknown and we have now found these motifs in a small family of plant enzymes, two of which are known to function in chlorophyll b and Gly betai...

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Flow cytometry of mesophyll and bundle sheath chloroplast thylakoids of maize (Zea mays L.)

Cited by 16 publications

References 29 publications

Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants

Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants

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

Light-Dependent Death of Maize lls1 Cells Is Mediated by Mature Chloroplasts

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Flow cytometry of mesophyll and bundle sheath chloroplast thylakoids of maize (Zea mays L.)

Cited by 16 publications

References 29 publications

Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants

Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants

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

Light-Dependent Death of Maize lls1 Cells Is Mediated by Mature Chloroplasts

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