Regulation of photosynthesis during heterocyst differentiation in Anabaena sp. strain PCC 7120 investigated in vivo at single-cell level by chlorophyll fluorescence kinetic microscopy.

Changes of photosynthetic activity in vivo of individual heterocysts and vegetative cells in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 during the course of diazotrophic acclimation were determined using fluorescence kinetic microscopy (FKM). Distinct phases of stress and acclimation following nitrogen step-down were observed. The first was a period of perception, in which the cells used their internally stored nitrogen without detectable loss of PS II activity or pigments. In the second, the stress phase of nitrogen limitation, the cell differentiation occurred and an abrupt decline of fluorescence yield was observed. This decline in fluorescence was not paralleled by a corresponding decline in photosynthetic pigment content and PS II activity. Both maximal quantum yield and sustained electron flow were not altered in vegetative cells, only in the forming heterocysts. The third, acclimation phase started first in the differentiating heterocysts with a recovery of PS II photochemical yields [Formula: see text] Afterwards, the onset of nitrogenase activity was observed, followed by the restoration of antenna pigments in the vegetative cells, but not in the heterocysts. Surprisingly, mature heterocysts were found to have an intact PS II as judged by photochemical yields, but a strongly reduced PS II-associated antenna as judged by decreased F 0. The possible importance of the functional PS II in heterocysts is discussed. Also, the FKM approach allowed to follow in vivo and evaluate the heterogeneity in photosynthetic performance among individual vegetative cells as well as heterocysts in the course of diazotrophic acclimation. Some cells along the filament (so-called "superbright cells") were observed to display transiently increased fluorescence yield, which apparently proceeded by apoptosis.

Traffic lights in trichodesmium. Regulation of photosynthesis for nitrogen fixation studied by chlorophyll fluorescence kinetic microscopy.

We investigated interactions between photosynthesis and nitrogen fixation in the non-heterocystous marine cyanobacterium Trichodesmium IMS101 at the single-cell level by two-dimensional (imaging) microscopic measurements of chlorophyll fluorescence kinetics. Nitrogen fixation was closely associated with the appearance of cells with high basic fluorescence yield (F(0)), termed bright cells. In cultures aerated with normal air, both nitrogen fixation and bright cells appeared in the middle of the light phase. In cultures aerated with 5% oxygen, both processes occurred at a low level throughout most of the day. Under 50% oxygen, nitrogen fixation commenced at the beginning of the light phase but declined soon afterwards. Rapid reversible switches between fluorescence levels were observed, which indicated that the elevated F(0) of the bright cells originates from reversible uncoupling of the photosystem II (PSII) antenna from the PSII reaction center. Two physiologically distinct types of bright cells were observed. Type I had about double F(0) compared to the normal F(0) in the dark phase and a PSII activity, measured as variable fluorescence (F(v) = F(m) - F(0)), similar to normal non-diazotrophic cells. Correlation of type I cells with nitrogen fixation, oxygen concentration, and light suggests that this physiological state is connected to an up-regulation of the Mehler reaction, resulting in oxygen consumption despite functional PSII. Type II cells had more than three times the normal F(0) and hardly any PSII activity measurable by variable fluorescence. They did not occur under low-oxygen concentrations, but appeared under high-oxygen levels outside the diazotrophic period, suggesting that this state represents a reaction to oxidative stress not necessarily connected to nitrogen fixation. In addition to the two high-fluorescence states, cells were observed to reversibly enter a low-fluorescence state. This occurred mainly after a cell went through its bright phase and may represent a fluorescence-quenching recovery phase.

Copper-induced inhibition of photosynthesis: limiting steps of in vivo copper chlorophyll formation in Scenedesmus quadricauda.

The in vivo substitution of Mg2+ in chlorophyll by heavy metals is an important damage mechanism in heavy metal-stressed plants that leads to an inhibition of photosynthesis. In photosynthetic organisms with LHC II antennae, the in vivo substitution of Mg2+ by Cu2+ occurs particularly readily under low irradiance with a dark phase - a phenomenon referred to as 'shade reaction'. In the present study the limiting steps of the shade reaction were investigated with synchronised cultures of the chlorococcal green alga Scenedesmus quadricauda (Turp.) Bréb. The rate of copper chlorophyll formation during shade reaction was shown to be controlled by several factors; firstly, in some phases of the cell cycle, especially at the end of the light period, Mg2+ in chlorophyll was not accessible to substitution. This pattern is likely to be caused by cell cycle-dependent changes in photosynthesis and thylakoid ultrastructure, which were published earlier and are reconsidered in the discussion of the present work. Secondly, prolonged culture in a medium containing 3 µM Cu2+ reversibly increased the resistance of the strain to Cu2+. Culturing without added Cu2+ lowered the threshold concentrations of various deleterious effects more than 10-fold within 8 months of de-adaptation. Adaptation to high Cu2+ levels is discussed in the context of studies of the regulation of metal transporter proteins. In addition, it was also observed that toxic Cu2+ levels impaired photosynthesis sooner than cell division.

New insights into photosynthetic oscillations revealed by two-dimensional microscopic measurements of chlorophyll fluorescence kinetics in intact leaves and isolated protoplasts.

Chlorophyll fluorescence kinetic microscopy was used to analyze photosynthetic oscillations in individual cells of leaves and in isolated leaf cell protoplasts. Four Brassicaceae species were used: Arabidopsis halleri (L.) O'Kane & Al-Shehbaz, Thlaspi fendleri (Nels.) Hitchc, Thlaspi caerulescens J.&C. Presl and Thlaspi ochroleucum Boiss et Helder. With the latter two, the measurements were extended also to isolated protoplasts. The oscillations were induced under the microscope by exposing dark-adapted samples to actinic irradiance. Detailed analysis of the induced transients revealed that they consist of several processes oscillating with different frequencies and not only one component as reported earlier. Furthermore, it was found that most of these processes are controlled inside each individual cell. This was shown by differences in oscillations in neighboring cells and protoplasts that share a uniform intercellular environment. The frequency of the dominant oscillation frequency depended neither on irradiance nor on CO2 concentration and is, therefore, not controlled by the photosynthetic rate. Characteristic differences in the frequency spectrum and damping of oscillations have been found among the plant species examined.