French bean (Phaseolus vulgaris L.) cotyledons lost most of their reserve substances during several early days of germination and turned green. In cotyledon mesophyll cells of one-week-old seedlings, plastids were represented predominantly by amyloplasts (starch grains) and chloroamyloplasts, and the cells appeared to be metabolically highly active. Cell heterogeneity associated with distance of the cells from cotyledon vascular bundles was evident. Only mesophyll cells near to the bundles were rich in plastids. In two-weeks-old intact bean plants, the cotyledons were yellow and shrunken, and their cells were nearly "empty". The plastids in them were represented by senescent plastids (gerontoplasts) only. In the gerontoplasts as well as freely in cytosol, fluorescent lipoid inclusions were accumulated. This cotyledon development was more or less independent of irradiance. In "decapitated" bean plants, senescence of mesophyll cells and plastids was slowed down considerably, and the life span of the cotyledons was prolonged. and J. Kutík, N. Wilhelmová, J. Snopek.
We exploited leaves of tobacco (Nicotiana tabacum L., cv. Wisconsin 38) with introduced chimeric construct consisting of SAG12 promoter fused with ipt gene for cytokinin synthesis and therefore prolonged life-span. As a control we used its wild type. In 12-week-old plants, the first leaves of control plants showed senescence symptoms at the time of sampling. Carotenoid content decreased with increasing leaf age both in control and in transgenic plants. On the other hand, the first leaves of transgenic plants demonstrated better antioxidant capacity represented by carotenoids compared to the leaves of control plants of the same age. They stayed still green at this age. and D. Procházková, D. Haisel, N. Wilhelmová.
The influence of a cytokinin, 6-benzylaminopurine (BAP), on chloroplast structure was studied using biochemical methods and electron microscopy. The average degree of thylakoid stacking was determined by digitonin fractionation and differential centrifugation of chloroplasts from tobacco plantlets after treatment with different concentrations of BAP in agar medium during cultivation in viíro. An elevated concentration of BAP in the medium induced an increase in grana stacking. This was in accordance with the lowering of the chlorophyll a/b ratio in these chloroplasts. The relative amoímt of proteins and carotenoids increased in both stromal and (to a lesser extent) granal chloroplast thylakoid fractions with the BAP concentration. The electron microscopic studies revealed nearly the same volume density of thylakoid membranes within chloroplasts of BAP treated plantlets and control ones. In the BAP treated plantlets the chloroplasts were smaller and had a profound accumulation of starch inclusions and a more flattened shape than the chloroplasts of control plantlets. The volume density of plastoglobuli in chloroplasts did not decrease under the influence of BAP,
The content of cytokinins (CKs), the plant inhibitors of the final phase of plant development, senescence, is effectively controlled by irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). In transgenic tobacco, denoted as AtCKX, with over-expressed CKX causing lowered CK content, we investigated changes in the time courses of chlorophyll (Chl) and xanthophyll (violaxanthin, antheraxanthin, zeaxanthin, neoxanthin, and lutein) contents. We also determined parameters of slow Chl fluorescence kinetics such as minimum Chl fluorescence yield in the darkadapted state F0, maximum quantum yield of PS2 photochemistry (Fv/Fm), maximum ratio of quantum yields of photochemical and concurrent non-photochemical processes in photosystem 2 (PS2), Fv/F0, non-photochemical quenching (NPQ), and effective quantum yield of photochemical energy conversion in PS2 (Φ2). We used three different developmental leaf stages, old, mature, and young, and compared this with time courses of these characteristics in leaves with natural CK levels. The parameters Fv/Fm, Fv/F0, and Φ2 were unchanged during ageing in AtCKX plants in contrast to control ones where a significant decrease in old leaves was found. In control plants F0 increased during ageing, but in the oldest leaf a considerable decrease was observed. This could indicate progressive damage to PS2 reaction centres and then detachment and rapid degradation of Chl. This is in agreement with time course of Chl content. NPQ decreased with age and was similar in both plant types. We observed a decline of xanthophyll contents in the oldest leaves in both plant types, but the contents were enhanced in AtCKX compared to control plants, especially of neoxanthin. The higher xanthophyll contents in the transgenic plants contribute to a better photoprotection and the fluorescence parameters indicated that photosynthetic apparatus was in better condition compared to control and it consequently postponed the onset of leaf senescence. and Z. Mýtinová, D. Haisel, N. Wilhelmová.
In tobacco leaves inoculated with tobacco mosaic virus (TMV), changes in chlorophyll (Chl) and carotenoid contents, parameters of slow Chl fluorescence kinetics, i.e. the maximum quantum yield of photosystem (PS2) photochemistry Fv/Fm, the effective quantum yield of photochemical energy conversion in PS2 Φ2, ratio of quantum yields of photochemical and concurrent non-photochemical processes in PS2 Fv/F0, non-photochemical quenching (NPQ), and photochemical activities of isolated chloroplasts from systemically infected tobacco leaves were investigated. We compared two successive stages of infection, the first in the stage of vein clearing at 9th day post inoculation (dpi) and the second at 22nd dpi when two different regions, i.e. light- (LGI) or dark-green (DGI) islands in the infected leaf were apparent and symptoms were fully developed. These two different regions were measured separately. The Chl and carotenoid contents in infected leaves decreased with a progression of infection and were lowest in LGI in the second stage. Also the ratio of Chl a/b declined in similar manner. The maximum quantum yield of PS2 photochemistry Fv/Fm, was decreased in the following order: first stage, DGI, and LGI. The same is true for the ratio Fv/F0. The decrease of Φ2 in infected leaves declined as compared to their controls. On the contrary, NPQ increased in infected leaves, the highest value was found in the first infection stage. Photochemical activities of the whole electron transport chain in isolated chloroplasts dramatically declined with the progression of symptoms, the lowest value was in LGI. Similarly, but to a lesser extent, the activity of PS2 in isolated chloroplasts decreased in infected leaves. Generally, the most marked impairment of the photosynthetic apparatus was manifested in the LGI of infected leaves. and N. Wilhelmová ... [et al.].
Ability of detergents digitonin (Dg), Triton X-IOO (TX), sodium dodecylsulphate (SDS), cetylpyridinium chloride (CPC), and Zwittergents 3-12 (Z-12), 3-14 (Z-14), and 3-16 (Z-16) to fragment unstacked thylakoid membranes was tested in French beán (Phaseolus vulgaris L.) leaves differing in age. The power of selected detergents to initiate fragmentation did not differ with different Chemical structure and molecular size of detergents, but it was controlled more probably by their molecular charge and shape. Unfavourable was the presence of positive charge and the absence of alkyl chain in detergent molecule. Membrane fragments obtained after the action of individual detergents on agranal thylakoids were separated on a Percoll gradient. Separated fractions were characterized by their chlorophyll (Chl) content, Chl a/b and Chl/protein ratios, position of absorption maximum (A^gx) i” spectral region and ratios of intensities of fluorescence emission bands. Since separation of no Chl-protein was achieved, no priority of detergent binding to any membrane component occurred under the conditions ušed. Fractions of similar density, products of the action of low concentrations of different detergents on the chloroplasts from leaves of the same age, differed in their characteristics. Low concentrations of detergents fragmented agranal membranes in a pattem which changed during leaf ontogeny. Only Z-16 had an increased capacity to extract proteins from membrane; this was probably promoted by the presence of 16-C alkyl chain.