Plastid redox state and sugars: interactive regulators of nuclear-encoded photosynthetic gene expression.

Feedback regulation of photosynthesis by carbon metabolites has long been recognized, but the underlying cellular mechanisms that control this process remain unclear. By using an Arabidopsis cell culture, we show that a block in photosynthetic electron flux prevents the increase in transcript levels of chlorophyll a/b-binding protein and the small subunit of Rubisco that typically occurs when intracellular sugar levels are depleted. In contrast, the expression of the nitrate reductase gene, which is induced by sugars, is not affected. These findings were confirmed in planta by using Arabidopsis carrying the firefly luciferase reporter gene fused to the plastocyanin and chlorophyll a/b-binding protein 2 gene promoters. Transcription from both promoters increases on carbohydrate depletion. Blocking photosynthetic electron transport with 3-(3', 4'-dichlorophenyl)-1,1'-dimethylurea prevents this increase in transcription. We conclude that plastid-derived redox signaling can override the sugar-regulated expression of nuclear-encoded photosynthetic genes. In the sugar-response mutant, sucrose uncoupled 6 (sun6), plastocyanin-firefly luciferase transcription actually increases in response to exogenous sucrose rather than decreasing as in the wild type. Interestingly, plastid-derived redox signals do not influence this defective pattern of sugar-regulated gene expression in the sun6 mutant. A model, which invokes a positive inducer originating from the photosynthetic electron transport chain, is proposed to explain the nature of the plastid-derived signal.

Photosynthetic light response curves determined with the leaf oxygen electrode: minimisation of errors and significance of the convexity term.

From photosynthetic studies on a range of monocotyledonous (C-3 and C-4) and dicotyledonous (C-3) plants using a leaf oxygen electrode, we conclude the following. (i) A non-linear model [J.H.M. Thornley (1976) Mathematical models in plant physiology, Academic Press, London; B. Marshall and P.V. Biscoe (1980) J Exp Bot 31:29-39] significantly better describes the photosynthetic light response curve [rate of photosynthesis (P) versus incident photosynthetic photon flux density (I)] than the frequently used linear hyperbolic model [E.I. Rabinowich (1951) Photosynthesis and related processes, vol 2, Wiley, New York]. (ii) When used at the recommended CO2 partial pressures (Ca = 1-5 kPa), CO2 supply saturates the photosynthesis rate in the C-3 dicot Phaseolus coccineus L. but not in the C-3 monocot Hordeum vulgare L.. (iii) Fits using a linear hyperbolic model for P versus I produce relatively large and statistically significant errors (approximately 60%) in the estimation of Pmax and quantum efficiency (alpha) if Ca is not > 5 kPa. (iv) The convexity term, theta, incorporated into the non-linear models for P versus I appears to reflect the limitation placed on the carboxylation processes by the supply of CO2 to the chloroplast stroma. Therefore, the use of a non-linear model providing an estimate of theta should be encouraged, as it is likely to provide information on the physiological status of plants.

Selective release, removal, and reconstitution of bacteriochlorophyll a molecules into the B800 sites of LH2 complexes from Rhodopseudomonas acidophila 10050.

A method is described which allows the selective release and removal of the Bchla-B800 molecules from the LH2 complex of Rhodopseudomonas acidophila 10050. This procedure also allows reconstitution of approximately 80% of the empty binding sites with native Bchla. As shown by circular dichroism spectroscopy, the overall structures of the B850-only and reconstituted complexes are not affected by the pigment-exchange procedure. The pigments reconstituted into the B800 sites can also efficiently transfer excitation energy to the Bchla-B850 molecules.

A kinetic model for subtractive hybridization.

Nucleic acid sequences that differ in abundance between two populations (target sequences) can be cloned by multiple rounds of subtractive hybridization and amplification by PCR. These sequences can be cDNAs representing up-regulated mRNAs, or genomic DNAs from deletion mutants. We have derived an equation that describes the recovery of such sequences, and have used this to simulate the outcome of up to 10 rounds of subtractive hybridization and PCR amplification. When the model was tested by comparing its predictions with the published results from genomic and cDNA subtractions, the predictions of the model were generally in good agreement with the published data. We have modelled the outcomes of genomic subtractions, for a variety of genomes, and have used it to compare various strategies for enriching targets. The model predicts that for genomes of less than 5 x 10(8) bp, deletions of as small as 1 kbp should represent > 99% of the DNA after three to six rounds of hybridization (depending on the enrichment procedure). As genomes increase in size, the kinetics of hybridization become an important limiting factor. However, even for genomes as large as 3 x 10(9) bp, it should be possible to isolate deletions of 5 kbp using the appropriate conditions. These simulations suggest that such methods offer a realistic alternative to chromosome walking for identifying genomic deletions for which there are known phenotypes, thereby considerably reducing time and effort. For cDNA subtractive hybridization, the model predicts that after six rounds of hybridization, sequences that do not differ in abundance between the tester and driver populations (the background) will represent < 1% of the subtracted population, and even quite modestly upregulated cDNAs should be successfully enriched. Where several up-regulated cDNAs are present, the predicted final representation is dependent on both the initial abundance and the degree of up-regulation.

State adaptations in the cyanobacterium Synechcoccus 6301 (PCC): Dependence on light intensity or spectral composition?

A profile of high light to intense self-shading conditions was constructed using a white light source and cultures of the cyanobacterium Synechococcus 6301; this profile approximates to a natural self-shading gradient of decreasing light intensity and PS II/PS I excitation ratio. Samples of S.6301 were placed along this profile and allowed to state adapt. To separate the effects of light intensity and wavelength on state adaptation, samples were also placed in a shade profile produced by a white light source and neutral density filters. After adaptation, samples were fixed in their resulting state by the addition of glutaraldehyde, and fluorescence measurements were made at 35° C or -160 °C. It is concluded: 1. Under conditions of deep shade (<5 µmol m(-2)s(-1) PAR) and weak shade (>200 µmol m(-2)s(-1) PAR), cells adapt to a low PS II fluorescence state (state 2); in moderate shade (20-60 µmol m(-2)s(-1)PAR) cells adapt to a high PS II fluorescence state (state 1). We suggest these findings provide evidence for the operation of different factors on the control of state adaptations in cyanobacteria; one set operates at low light and another at high light intensities. 2. Under conditions of self-shading, there is little evidence to support the contention that state adaptations in cyanobacteria are produced by wavelength-dependent changes in the PS II/PS I excitation ratio, instead, it appaers they are produced by changes in the intensity of incident irradiation. 3. The observed fluorescence changes do not appear to involve major changes in the phycobilisome sensitisation of PS II and PS I. Instead, it appears that these changes are effected by alterations in Φ(F) of PS II (i.e. changes in PS II excitation density caused by alterations in the rate constants controlling spillover to PS I, photochemistry, fluorescence emission or thermal deactivation.

Identification of genes up-regulated in dedifferentiating Nicotania glauca pith tissue, using an improved method for constructing a subtractive cDNA library.

Pith explants of Nicotiana glauca grown in vitro in synthetic medium supplemented with 2,4 dichlorophenoxyacetic acid (2, 4 D), are induced to dedifferentiate. Treatment with actinomycin D within the first 4-8 h of culture (but not later) is lethal and the explants die, implying a requirement for de novo transcription. The genes expressed during the initial period of culture are presumably critical for subsequent cell survival and proliferation, but so far their identity is unknown. We have constructed a subtractive cDNA library, enriched in sequences more abundant in dedifferentiating tissue than in pith. The subtractive library contains approximately seven major species, two of which, NGSUB7 and NGSUB8, are highly abundant. In Northern blots, these two hybridized to mRNA species whose abundance increased significantly but transiently during the first 4 to 8 h of culture. The sequence of NGSUB7 showed no significant homology at a nucleotide or derived amino acid level with any previously reported sequence. NGSUB8 however, showed significant homology over part of the derived amino acid sequence to several yeast and bacterial proteins with DNA binding function. We propose that the two recombinants represent transcripts from two novel genes edeA and edeB, which are expressed early in dedifferentiation.

Inhibition of the k-stimulated ATPase of the plasmalemma of pinto bean leaves by ozone.

Three varieties of Phaseolus vulgaris which differ in their sensitivity to ozone were examined for changes in some physiological and structural plasma membrane characteristics. Plasma membrane vesicles were prepared from control and ozone-treated (0.2 to 0.5 microliters per liter ozone for 5 hours) leaf tissue, and the (K(+) + Mg(2+))-ATPase activity determined and compared. No major changes were observed in the resistant varieties. The sensitive variety showed a severe inhibition of ATPase activity which was largely due to a decrease in the K(+)-stimulated component. This inhibition was completely reversed by the addition of sulfhydryl compounds.Ozone-induced plasma membrane permeability changes may be effected by damage to membrane proteins, perhaps by oxidation of amino acid sulfhydryl groups to disulfide and sulfenic moieties.