Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L

The activities of chlorophyllase, contents of pigments including chlorophyll a and b, chlorophyllide a and b, and phaeophorbide a during leaf senescence under low oxygen (0.5% O2) and control (air) were investigated in a non-yellowing mutant and wild-type leaves of snap beans (Phaseolus vulgaris L.). Chlorophyllase from leaf tissues had maximum activity when incubated at 40C in a mixture containing 50% acetone. In both mutant and wild type, chlorophyllase activity was the highest in freshly harvested non-senescent leaves and decreased sharply in the course of senescence, indicating that the loss of chlorophylls in senescing leaves is not directly related to the activity of chlorophyllase and that chlorophyllase activity is not altered in the mutant. The wild type had higher ratios of chlorophyll a to chlorophyll b than the mutant and chlorophyll a : b ratios increased during senescence in both types. In the senescent mutant leaves, accumulations of chlorophyllide a and chlorophyllide b were detected, but no phaeophorbide a was found. Chlorophyllide b had a greater accumulation than chlorophyllide a in the early stage of senescence. Low oxygen treatment not only delayed chlorophyll degradation but also enhanced the accumulations of chlorophyllide a and b and lowered the ratios of chlorophyll a to chlorophyll b.     

Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees

The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R _Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves). Sun leaves exhibited higher photosynthetic rates P _N on a leaf area basis (mean of 9.1–10.1 μmol CO₂ m⁻² s⁻¹) and Chl basis, which correlated well with the higher values of stomatal conductance G _s (range 105–180 mmol m⁻² s⁻¹), as compared to shade leaves (G _s range 25–77 mmol m⁻² s⁻¹; P _N: 3.2–3.7 μmol CO₂ m⁻² s⁻¹). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R _Fd, which possessed higher values in sun leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In addition, via R _Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves.     

Relative binding affinities of chlorophylls in peridinin–chlorophyll–protein reconstituted with heterochlorophyllous mixtures

Peridinin–chlorophyll–protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which to study energy transfer among monomeric chlorophylls (Chls) by both bulk and single-molecule spectroscopy. They can be obtained by reconstituting the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization of these “half-mers”, homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers of native PCP from Amphidinium carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a.     

Characterization of chlorophyll–protein complexes isolated from a Siphonous green alga, Bryopsis corticulans

Six chlorophyll–protein complexes are isolated from thylakoid membranes of Bryopsis corticulans by dodecyl-β-d-maltoside polyacrylamide gel electrophoresis. Unlike that of higher plants, the 77 K fluorescence emission spectrum of the CP1 band, the PSI core complexes of B. corticulans, presents two peaks, one at 675 nm and the other at 715–717 nm. The emission peak at 715–717 nm is slightly higher than that at 675 nm in the CP1 band when excited at 438 or 540 nm. However, the peak at 715 nm is obviously lower than that at 675 nm when excited at 480 nm. The excitation spectra of CP1 demonstrate that the peak at 675 nm is mainly attributed to energy from Chl b while it is the energy from Chl a that plays an important role in exciting the peak at 715–717 nm. Siphonaxanthin is found to contribute to both the 675 nm and 715–717 nm peaks. We propose from the above results that chlorophyll a and siphonaxanthin are mainly responsible for the transfer of energy to the far-red region of PSI while it is Chl b that contributes most of the transfer of energy to the red region of PSI. The analysis of chlorophyll composition and spectral characteristics of LHCP¹ and LHCP³ also indicate that higher content of Chl b and siphonaxanthin, mainly presented in LHCP¹, the trimeric form of LHCII, are evolved by B. corticulans to absorb an appropriate amount of light energy so as to adapt to their natural habitats.     

Physiological,biochemical, and fluorescence parameters of senescing sugar beet leaves in the vegetative phase of growth

The phase of vegetative growth of sugar beet (Beta vulgaris L., single-sprout form) was conditionally subdivided into four periods according to leaf number and size (including already withered leaves): (A) 8 ± 1 weeks after seedling emergence (wase) (5–7 leaves); (B) 11 ± 1 wase (10–12 leaves); (C) 14 wase (13–15 leaves); (D) 15 wase (15–18 leaves). It took each next leaf about 1 week to come into view. In the course of leaf senescence, palisade parenchyma became less ordered; cells, vacuoles, and intercellular spaces expanded; leaf area and thickness increased. Chloroplasts became swollen, starch grains (and later osmiophilic globules) accumulated and degraded. In every growth period, the highest levels of soluble carbohydrates (sCH), chlorophyll (Chl (a + b)), soluble protein, and the highest activities of rubisco and soluble carboanhydrase usually preceded the full leaf expansion. In unfolded leaves at the growth period B, the maximum values of biochemical characteristics were as a rule higher than at the growth periods A and C and especially D. The only exception was Chl (a + b) with its peak level somewhat increased with plant age. Occurrence of peak values of individual characteristics depended on plant growth period. These characteristics started diminishing asynchronously, with a minimum in old operational leaves. Only the sCH content in the leaves at the periods C and D was stable. Changes in quantum yield at PSII and nonphotochemical fluorescence quenching reflected the age-associated differences in biochemical characteristics. The results are discussed in the light of the idea that leaf senescence is a normal stage of development directly related to the changes in source-sink relations. Biochemically, this stage comprises the degradation of temporarily stored products and partial utilization of the breakdown products for maintenance of the growth of newly formed leaves and root.     

Zinc-Induced Changes in Morpho-Physiological and Biochemical Parameters in Artemisia annua

Responses of Artemisia annua to different concentrations of zinc [50, 100, 200, 300 and 400 μg g⁻¹(soil dry mass)] were studied during plant ontogeny. Total leaf area, dry mass of leaves, length and dry mass of shoots and roots increased with the age of the plant but the magnitude of increase declined significantly under the influence of Zn treatment. Net photosynthetic rate, intercellular carbon dioxide concentration and stomatal conductance were highest at flowering stage in control and treated plants and decreased at post flowering stage. Contents of chlorophyll a, chlorophyll b, carotenoids, proteins and nitrate reductase activity in leaves increased from pre-flowering to maximum level at flowering stage and decreased thereafter in both control and treated plants. Presence of Zn in the soil drastically decreased/inhibited all the parameters, and the magnitude of decline increased with increasing Zn concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Conversion of chlorophyll b to chlorophyll a precedes magnesium dechelation for protection against necrosis in Arabidopsis

Chlorophyll is a deleterious molecule that generates reactive oxygen species and must be converted to non‐toxic molecules during plant senescence. The degradation pathway of chlorophyll a has been determined; however, that of chlorophyll b is poorly understood, and multiple pathways of chlorophyll b degradation have been proposed. In this study, we found that chlorophyll b is degraded by a single pathway, and elucidated the importance of this pathway in avoiding cell death. In order to determine the chlorophyll degradation pathway, we first examined the substrate specificity of 7‐hydroxymethyl chlorophyll a reductase. 7‐hydroxymethyl chlorophyll a reductase reduces 7‐hydroxymethyl chlorophyll a but not 7‐hydroxymethyl pheophytin a or 7‐hydroxymethyl pheophorbide a. These results indicate that the first step of chlorophyll b degradation is its conversion to 7‐hydroxymethyl chlorophyll a by chlorophyll b reductase, although chlorophyll b reductase has broad substrate specificity. In vitro experiments showed that chlorophyll b reductase converted all of the chlorophyll b in the light‐harvesting chlorophyll a/b protein complex to 7‐hydroxymethyl chlorophyll a, but did not completely convert chlorophyll b in the core antenna complexes. When plants whose core antennae contained chlorophyll b were incubated in the dark, chlorophyll b was not properly degraded, and the accumulation of 7‐hydroxymethyl pheophorbide a and pheophorbide b resulted in cell death. This result indicates that chlorophyll b is not properly degraded when it exists in core antenna complexes. Based on these results, we discuss the importance of the proper degradation of chlorophyll b.     

Chlorophyll/nutrient characteristics in the water masses to the north of South Georgia, Southern Ocean

Chlorophyll a and nutrient concentrations along with temperature and salinity values were measured at 22 CTD stations along a 735-km transect running to the northwest of the island of South Georgia, Southern Ocean. Measurements were repeated during five summer surveys (January and February 1994, January 1996, December 1996, January 1998) and one spring survey (October 1997). The transect sampled Sub-Antarctic Zone water in the north, Polar Frontal Zone water and Antarctic Zone water in the south. Chlorophyll a concentrations were lowest to the north of the transect and frequently high (up to 17 mg m⁻³) in the deep open ocean of the Antarctic Zone. Sub-surface peaks were measured in all zones and chlorophyll a was detectable to a depth of 150 m. There was a clear latitudinal temperature gradient in the near-surface waters (0–50 m), the warmest water occurring in the north (∼12 °C), and the coolest in the Antarctic Zone (∼2 °C). There was also a well-defined latitudinal gradient in summer near-surface silicate concentrations (∼2, 4, and 10 mmol m⁻³ in the Sub-Antarctic Zone, the Polar Frontal Zone and the Antarctic Zone, respectively), increasing to >20 mmol m⁻³ near South Georgia. Distinct differences in silicate concentrations were also evident in all three zones to a depth of 500 m. Near-surface nitrate and phosphate concentrations were relatively low to the north of the transect (∼14 and 1 mmol m⁻³, respectively) and higher in the Polar Frontal Zone and Antarctic Zone (∼18 and 1.4 mmol m⁻³, respectively). Ammonium and nitrite were restricted to the upper 200 m of the water column, and exhibited sub-surface concentration peaks, the lowest being in the Sub-Antarctic Zone (0.68 and 0.25 mmol m⁻³, respectively) and the highest in the Antarctic Zone (1.72 and 0.29 mmol m⁻³, respectively). Surface (∼6 m) spring nutrient measurements provided an indication of pre-bloom conditions; ammonium and nitrite concentrations were low (∼0.27 and 0.28 mmol m⁻³, respectively), while silicate, nitrate and phosphate concentrations were high and similar to previously measured winter values (e.g. ∼26, 23, 2 mmol m⁻³, respectively in the Antarctic Zone). Although the values measured were very variable, and there was some evidence of a seasonal growth progression, the chlorophyll a and nutrient distribution patterns were dominated by intercruise (interannual) factors. Approximate nutrient depletions (spring minus summer) appeared similar in the Polar Frontal Zone and Antarctic Zone for nitrate and phosphate, while silicate showed a marked latitudinal increase from north to south throughout the transect. Highest chlorophyll a concentrations coincided with the highest apparent silicate depletions over the deep ocean of the Antarctic Zone. In this area, relatively warm, easterly flowing Antarctic Circumpolar Current water meets cooler, westerly flowing water that is influenced by the Weddell-Scotia Confluence and is rich in nutrients, especially silicate. Accepted: 27 November 1999     

Pigment dynamics and autumn leaf senescence in a New England deciduous forest,eastern USA

The leaves of woody plants at Harvard Forest in Central Massachusetts, USA, changed color during senescence; 70% (62/89) of the woody species examined anatomically contained anthocyanins during senescence. Anthocyanins were not present in summer green leaves, and appeared primarily in the vacuoles of palisade parenchyma cells. Yellow coloration was a result of the unmasking of xanthophyll pigments in senescing chloroplasts. In nine red-senescing species, anthocyanins were not detectable in mature leaves, and were synthesized de novo in senescence, with less than 20µg cm^–2 of chlorophyll remaining. Xanthophyll concentrations declined in relation to chlorophyll to the same extent in both yellow- and red-leaved taxa. Declines in the maximum photosystemII quantum yield of leaves collected prior to dawn were only slightly less in the red-senescing species, indicating no long-term protective activity. Red-leaved species had significantly greater mass/area and lower chlorophylla/b ratios during senescence. Nitrogen tissue concentrations in mature and senescent leaves negatively correlated to anthocyanin concentrations in senescent leaves, weak evidence for more efficient nitrogen resorption in anthocyanic species. Shading retarded both chlorophyll loss and anthocyanin production in Cornus alternifolia, Acer rubrum, Acer saccharum, Quercus rubra and Viburnum alnifolium. It promoted chlorophyll loss in yellow-senescing Fagus grandifolia. A reduced red:far-red ratio did not affect this process. Anthocyanins did not increase leaf temperatures in Q.rubra and Vaccinium corymbosum on cold and sunny days. The timing of leaf-fall was remarkably constant from year to year, and the order of senescence of individual species was consistent.     

Gradient of photosynthetic pigments in the bark and leaves of lilac (Syringa vulgaris L.)

The concentration of chlorophyll and a carotenoids in the bark of stems of different age and in the leaves of lilac (Syringa vulgaris L.) was determined. The thickness of bark changes with the age of the stems, ranging from 0.73 mm in the current-year stems to 1.22 mm in 3-year-old ones. Chlorophyll and carotenoids were present through the whole thickness of the bark, except the cork. It was found that chlorophyll and carotenoids are located mainly in the outer layer of the bark, immediately under the cork, to a depth of 400 μm. In this layer the chlorophyll a/b ratio is the highest and the content of chlorophyll is four times larger than that of carotenoids. When penetrating deeper into the bark, the content of chlorophyll and carotenoids as well as the chlorophyll a/b ratio diminishes. Investigations of the leaves showed that most of the chlorophyll is found in the palisade parenchyma, the chlorophyll a/b ratio is the highest in the upper layer. The highest concentration of chlorophyll in the bark is 0.44 mg·dm⁻² and in leaves −1.2 mg⁻²·dm⁻². The highest value of the chlorophyll a/b ratio in the bark is 3.8, and the lowest 0.5, while in the leaves it varies from 4.5 to 3.8 Low values of the chlorophyll a/b ratio are due to the shade conditions existing in the bark and they are evidence of very great differentiation of light conditions within it.     

Phytoplankton and particulate matter at the Weddell / Scotia Confluence (47°W) in summer 1989, as a final step of a temporal succession (EPOS project)

During January 1989, phytoplankton biomass and species composition were studied in a north / south transect at the Weddell / Scotia Confluence (47°W), between 57° and 61°30′S. Results showed a diatom bloom in the Scotia Sea (chlorophyll a 1.9 μg l⁻¹, particulate organic carbon 239 μg l⁻¹), dominated by Fragilariopsis cylindrus, Dactyliosolen antarcticus and Chaetoceros dichaeta. Low chlorophyll a / phaeopigments ratios (about 1.4) and silicate concentrations (15 μmol l⁻¹) suggested that this was an advanced bloom phase, probably linked to high grazing pressure. Minimum chlorophyll a values of 0.1–0.2 μg l⁻¹ and particulate organic carbon 46 μg l⁻¹ were found at the Weddell / Scotia Front and in a subsurface layer of the Weddell Sea Water. In the southern part of the transect (61°30′S), in the Weddell Sea, a second surface maximum was found (chlorophyll a 0.9 μg l⁻¹, particulate organic carbon 120 μg l⁻¹), but with a different species composition, with Cryptomonas sp. dominant. Our results show a succession within the diatom community in the Weddell / Scotia Confluence Waters when comparing the three EPOS legs. In the Weddell Sea from spring to summer, nanoflagellates, with only a minor contribution from diatoms, persist over a long period with little change in the community structure. We suggest that the frontal system, together with the receding ice edge and the grazing pressure of either krill or protozooplankton, are mainly responsible for the phytoplankton distribution patterns found. Received: 3 July 1996 / Accepted: 3 November 1996     

Excess irradiance causes early symptoms of senescence during leaf expansion in photoautotrophically <Emphasis Type="Italic">in vitro</Emphasis> grown tobacco plants

Photosynthetic parameters, growth, and pigment contents were determined during expansion of the fourth leaf of in vitro photoautotrophically cultured Nicotiana tabacum L. plants at three irradiances [photosynthetically active radiation (400–700 nm): low, LI 60 μmol m⁻² s⁻¹; middle, MI 180 μmol m⁻² s⁻¹; and high, HI 270 μmol m⁻² s⁻¹]. During leaf expansion, several symptoms usually accompanying leaf senescence appeared very early in HI and then in MI plants. Symptoms of senescence in developing leaves were: decreasing chlorophyll (Chl) a+b content and Chl a/b ratio, decreasing both maximum (F_V/F_M) and actual (Φ_PS2) photochemical efficiency of photosystem 2, and increasing non-photochemical quenching. Nevertheless, net photosynthetic oxygen evolution rate (P _N) did not decrease consistently with decrease in Chl content, but exhibited a typical ontogenetic course with gradual increase. P _N reached its maximum before full leaf expansion and then tended to decline. Thus excess irradiance during in vitro cultivation did not cause early start of leaf senescence, but impaired photosynthetic performance and Chl content in leaves and changed their typical ontogenetic course.     

Photosynthetic Characteristics of Chloroplasts of Primary Wheat Leaves Grown under Different Irradiance

The rate of accumulation of total chlorophyll (Chl) and carotenoids (Car) of leaves grown under high irradiance, HI (30 and 45 W m^–2) was faster than at moderate irradiance, MI (15 W m^–2). However, the senescence phase started earlier in the samples and proceeded at a faster rate. Chl a/b and Chl (a+b)/Car values showed faster loss of Chl a (compared to Chl b) and Chl (a+b) (compared to Car) in HI leaves. Protein accumulation and loss were also similar to that of Chl (a+b) content. Increase in Chl fluorescence during the development phase may suggest a gradual change in thylakoid organisation, however, the temporal kinetics were different in HI and MI samples. Increase in fluorescence polarisation during senescence of HI leaves compared to the control (MI) suggests conversion of thylakoid membranes to gel phase. Chloroplasts prepared from HI seedlings showed higher rate of photochemical activities, however, the activity declined earlier and at faster rate compared to the control.     

Optical fractionation of chlorophyll and primary production for coastal waters of the Southern Ocean

Our objective was to quantify the potential variability in remotely sensed chlorophyll a (Chl a) and primary productivity in coastal waters of the Southern Ocean. From data collected throughout the springs/summers of 1991–1994, we calculated the proportion of water column Chl a and primary productivity within the upper optical attenuation length (K ⁻¹ _par) and the satellite-weighted depth. The temporal variability was resolved every 2–3 days and was observed to be greater within years than between years. Three-year averages (n=223) revealed that 10.2 ± 3.6% of total Chl a and 14.8 ± 6.5% of production occurred within satellite-weighted depth in predominantly Case I waters. The average values were twice as high within K ⁻¹ _par, 24.1 ± 8% of total Chl a and 34 ± 9% of production respectively. Masked in these long-term averages are very large changes occurring on short time scales of seasonal blooms. We observed that the patterns of Chl a vertical distribution within blooms are also subject to taxonomic influence and dependent upon the physiological state of the phytoplankton. Highest proportions of water column Chl a in the first optical depth were measured during the rapid onset of surface cryptophyte blooms each year, i.e. 50% within K ⁻¹ _par and 30% above the satellite-weighted depth. Lowest fractions, 6% and 2% of biomass within K ⁻¹ _par and satellite-weighted depth respectively, were associated with peak bloom conditions independent of taxonomy. Our analyses suggest that satellite-dependent models of Chl a and subsequent chlorophyll-dependent primary production will be challenging to develop for the near-shore Southern Ocean, especially given the potentially high natural variability in the vertical distribution of Chl a driven by physical forcing, the photoadaptive abilities of polar phytoplankton, and taxonomic influences. Accepted: 27 August 1999     

Vertical distribution of Microcystis population in the regulated Nakdong River, Korea

The vertical distribution of a bloom-forming Microcystis population was studied based on the relevant limnological parameters obtained from the lower Nakdong River (Mulgum) during the summer of 1994. Over three months (late June to late September), a high abundance of Microcystis population (mean ± SD, 2.9 ± 8.4 × 10⁵ cells ml⁻¹, n = 40) and algal biomass (mean ± SD, chlorophyll a, 131 ± 346 μg l⁻¹, n = 31) was persistent throughout the entire water column (0–5 m, n = 11). The vertical distribution of carbon content was uneven, with a high concentration near the surface zone (mean ± SD, total, 7.9 ± 7.8; Microcystis, 5.2 ± 8.3 μg C ml⁻¹, n = 15). Incorporating limnological and meteorological factors, a diel study of the vertical distribution of Microcystis showed that the chlorophyll a concentration was highest near the surface zone on a calm night (wind velocity, <2 m s⁻¹, 2300–700) but was evenly distributed on a windy day (>4 m s⁻¹, 1100–1900). Among many possible factors, wind velocity may have played an important role in controlling the vertical distribution of Microcystis in the lower Nakdong River. Received: July 12, 1999 / Accepted: November 15, 1999     

The Relative Rates of Thiol–Thioester Exchange and Hydrolysis for Alkyl and Aryl Thioalkanoates in Water

This article reports rate constants for thiol–thioester exchange (k _ex), and for acid-mediated (k _a), base-mediated (k _b), and pH-independent (k _w) hydrolysis of S-methyl thioacetate and S-phenyl 5-dimethylamino-5-oxo-thiopentanoate—model alkyl and aryl thioalkanoates, respectively—in water. Reactions such as thiol–thioester exchange or aminolysis could have generated molecular complexity on early Earth, but for thioesters to have played important roles in the origin of life, constructive reactions would have needed to compete effectively with hydrolysis under prebiotic conditions. Knowledge of the kinetics of competition between exchange and hydrolysis is also useful in the optimization of systems where exchange is used in applications such as self-assembly or reversible binding. For the alkyl thioester S-methyl thioacetate, which has been synthesized in simulated prebiotic hydrothermal vents, k _a = 1.5 × 10⁻⁵ M⁻¹ s⁻¹, k _b = 1.6 × 10⁻¹ M⁻¹ s⁻¹, and k _w = 3.6 × 10⁻⁸ s⁻¹. At pH 7 and 23°C, the half-life for hydrolysis is 155 days. The second-order rate constant for thiol–thioester exchange between S-methyl thioacetate and 2-sulfonatoethanethiolate is k _ex = 1.7 M⁻¹ s⁻¹. At pH 7 and 23°C, with [R″S(H)] = 1 mM, the half-life of the exchange reaction is 38 h. These results confirm that conditions (pH, temperature, pK _a of the thiol) exist where prebiotically relevant thioesters can survive hydrolysis in water for long periods of time and rates of thiol–thioester exchange exceed those of hydrolysis by several orders of magnitude.     

Involvement of AtNAP1 in the regulation of chlorophyll degradation in Arabidopsis thaliana

In plants, chlorophyll is actively synthesized from glutamate in the developmental phase and is degraded into non-fluorescent chlorophyll catabolites during senescence. The chlorophyll metabolism must be strictly regulated because chlorophylls and their intermediate molecules generate reactive oxygen species. Many mechanisms have been proposed for the regulation of chlorophyll synthesis including gene expression, protein stability, and feedback inhibition. However, information on the regulation of chlorophyll degradation is limited. The conversion of chlorophyll b to chlorophyll a is the first step of chlorophyll degradation. In order to understand the regulatory mechanism of this reaction, we isolated a mutant which accumulates 7-hydroxymethyl chlorophyll a (HMChl), an intermediate molecule of chlorophyll b to chlorophyll a conversion, and designated the mutant hmc1. In addition to HMChl, hmc1 accumulated pheophorbide a, a chlorophyll degradation product, when chlorophyll degradation was induced by dark incubation. These results indicate that the activities of HMChl reductase (HAR) and pheophorbide a oxygenase (PaO) are simultaneously down-regulated in this mutant. We identified a mutation in the AtNAP1 gene, which encodes a subunit of the complex for iron–sulfur cluster formation. HAR and PaO use ferredoxin as a reducing power and PaO has an iron-sulfur center; however, there were no distinct differences in the protein levels of ferredoxin and PaO between wild type and hmc1. The concerted regulation of chlorophyll degradation is discussed in relation to the function of AtNAP1.     

Influence of water activity and temperature on in vitro growth of surface cultures of a Phoma sp. and production of the pharmaceutical metabolites, squalestatins S1 and S2

A Phoma sp., known to produce the pharmaceutically active metabolites squalestatin 1 (S1) and squalestatin 2 (S2), was cultured on malt-extract/agar (MEA) over a range of water activities (a _w, 0.995–0.90) and temperatures (10–35 °C) to investigate the influence on growth and metabolite production. Use of the ionic solute NaCl to adjust a _w resulted in significantly lower (P < 0.01) squalestatin yields than when the Phoma sp. was grown on MEA amended with the non-ionic solute glycerol. Water activity and temperature and their interactions were highly significant factors (P < 0.001) affecting growth of the Phoma sp., with optimum conditions of 0.998–0.980 a _w and 25 °C. Squalestatin production was similarly influenced by a _w, temperature, time and their interactions (P < 0.001). S1 and S2 production occurred over a narrower a _w and temperature range than growth, with a slightly lower optimum a _w range of 0.995–0.980 a _w. The optimum temperature for squalestatin production varied from 20 °C (S1) to 25 °C (S2) and yields of S2 were up to 1000 times lower than those of S1. The ratio of S1 and S2 produced by the Phoma sp. was influenced by a _w and temperature, with highest values at 0.99–0.98 a _w, and at 15 °C. Incubation times of 28 days gave highest yields of both S1 and S2. Up to 2000-fold increases in squalestatin yields were measured at optimum environmental conditions, compared to the unmodified MEA. This indicates the need to consider such factors in screening systems used to detect biologically active lead compounds produced by fungi. Received: 2 June 1997 / Received last revision: 6 November 1997 / Accepted: 7 November 1997     

The development of antenna complexes of barley (Hordeum vulgare cv. Akcent) under different light conditions as judged from the analysis of 77 K chlorophyll a fluorescence spectra

Using 77 K chlorophyll a (Chl a) fluorescence spectra in vivo, the development was studied of Photosystems II (PS II) and I (PS I) during greening of barley under intermittent light followed by continuous light at low (LI, 50 μmol m⁻² s⁻¹) and high (HI, 1000 μmol m⁻² s⁻¹) irradiances. The greening at HI intermittent light was accompanied with significantly reduced fluorescence intensity from Chl b excitation for both PS II (F685) and PS I (F743), in comparison with LI plants, indicating that assembly of light-harvesting complexes (LHC) of both photosystems was affected to a similar degree. During greening at continuous HI, a slower increase of emission from Chl b excitation in PS II as compared with PS I was observed, indicating a preferred reduction in the accumulation of LHC II. The following characteristics of 77 K Chl a fluorescence spectra documented the photoprotective function of an elevated content of carotenoids in HI leaves: (1) a pronounced suppression of Soret region of excitation spectra (410–450 nm) in comparison with the red region (670–690 nm) during the early stage of greening indicated a strongly reduced excitation energy transfer from carotenoids to the Chl a fluorescing forms within PS I and PS II; (2) changes in the shape of the excitation band of Chl b and carotenoids (460–490 nm) during greening under continuous light confirmed that the energy transfer from carotenoids to Chl a within PS II remained lower as compared with the LI plants. This revised version was published online in June 2006 with corrections to the Cover Date.