Supply of nitrogen can reverse senescence processes and affect expression of genes coding for plastidic glutamine synthetase and lysine-ketoglutarate reductase/saccharopine dehydrogenase.

Nitrogen availability has a strong influence on developmental processes in plants. We show that the time of nitrogen supply regulates the course of leaf senescence in flag leaves of Hordeum vulgare. The senescence-specific decrease in chlorophyll content and photosystem II efficiency is clearly delayed when plants are fertilised with nitrate at the onset of leaf senescence. Concurrently, the additional supply of nitrate affects expression patterns of two marker genes of nitrogen metabolism. As shown by quantitative RT-PCR analyses, senescence-specific downregulation of plastidic glutamine synthetase (GS2) and senescence-specific upregulation of lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) are both clearly retarded. Depletion of nitrogen in experiments using hydroponic growth systems results in premature primary leaf senescence. The already started senescence processes can be even reversed by later nitrogen addition, as proved by a further increase in photosystem II efficiency and chlorophyll content, returning to the high values of controls which had not been deprived of nitrogen. Although both addition of nitrate or ammonium effectively reversed nitrogen depletion-induced primary leaf senescence, addition of urea did not. Additionally, effects of nitrogen supply on the course of leaf senescence were analysed in the model plant Arabidopsis thaliana. Leaves of A. thaliana show the same reversion of senescence processes after receiving additional nitrogen supply, indicating that the nitrogen response of leaf development is conserved in different plant species.

Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves.

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.

Isolation and characterization of a Tritordeum cDNA encoding S-adenosylmethionine decarboxylase that is circadian-clock-regulated.

Sequence analysis of the two cDNA clones 47/11 and 50A which were isolated by differential screening of an explant cDNA library obtained from the monocot Tritordeum (hexaploid hybrid of diploid wild barley and tetraploid wheat lines) reveals that both clones include the same open reading frame (ORF). The sequence of this ORF shows a high degree of similarity with dicot S-adenosylmethionine decarboxylase (SAMDC) gene sequences and contains regions highly conserved in all known SAMDC sequences. It is further shown that the sequence represented by the cDNA clones 47/11 and 50A is derived from the wild barley (Hordeum chilense) genome, where it is present as a single-copy gene. Northern analyses indicate the corresponding transcript to accumulate in response to wounding and the transcript level changes with a circadian rhythm, having a beak in the middle of the light period. The periodicity continues in constant light, but is changed in constant darkness.

Expression of Early Light-Inducible Proteins in Flag Leaves of Field-Grown Barley.

Early light-inducible protein (ELIP) mRNA and protein levels were analyzed during maturation and senescence of barley (Hordeum vulgare L.) flag leaves under field conditions. The data clearly demonstrate that ELIP mRNA levels are related to the sunlight intensity before sample collection. Levels of mRNAs encoding both low and high molecular mass ELIPs fluctuate in parallel. Changes in mRNA levels are accompanied by corresponding changes in protein levels except for days when average temperatures are high. Comparison of flag leaves at different stages of development in spring and winter barley varieties suggests that light-stress-regulated ELIP gene expression is independent of the developmental stage of the leaves. Although chlorophyll content, photosystem II (PSII) efficiency, and 32-kD herbicide-binding protein of PSII levels decrease drastically after the onset of senescence, ELIP mRNA and protein still accumulate to high levels on bright days.

Photoisomerization of lycopene during carotenogenesis in mutant C-6D of Scenedesmus obliquus.

Mutant C-6D of the unicellular green alga Scenedesmus obliquus has lost the ability to form cyclic carotenoids during heterotrophic growth in the dark. In the dark it accumulates acyclic intermediates, i.e. lycopene, neurosporene and ζ-carotene. The lycopene and two neurosporene forms were identified to be cis-isomers. Upon transfer to light, intermediates decrease and a normal set of carotenoids is synthesized. Inhibition of the cyclization reaction by nicotine reveals a lightinduced isomerization of cis-lycopene to trans-lycopene. Since the spectral characteristics of these two isomers differ drastically the isomerization can be followed in vivo by measuring a light-induced absorbance change. This absorbance change has its maximum at 520 nm and shows fast kinetics under high light intensities reaching a saturation level after about 2 min. Fluence-response curves for this absorbance change were performed for different wavelengths of actinic light. From the linear parts of these curves an action spectrum was caculated showing maxima at 670, 630 and 440 nm originating from chlorophyll and a maximum at shorter wavelengths (400-510 nm) which is interpreted to derive from ζ-carotene. A model for the light regulation of carotenogenesis in mutant C-6D is presented and the relation to the so-called 520-change observed in many plants is discussed.

Relationship between biosynthesis of carotenoids and increasing complexity of photosystem I in mutant C-6D of Scenedesmus obtiquus.

Dark-grown cells of the pigment mutant C-6D of Scenedesmus obliquus, strain D3 (Gaffron 1939), contain only chlorophyll (Chl) a and carotenoid precursors. In these cells a functioning photosystem I (PSI) of basic structure was characterised by a high PSI activity and a low Chl/P700 ratio. The reaction-center complex of PSI (CPI) was shown to exist in the dark-grown cells. These findings demonstrate that the assembly of the core complex of PSI and its function are independent of the presence of carotenoids. Upon illumination, carotenoids, Ch1 b and additional Chl a were synthesized. Newly formed ß-carotene was shown by pigment analysis using high-performance liquid chromatography (HPLC) to be incorporated into CPI. Parallel to this process a shift of the long-wavelength fluorescence emission of PSI from 712-714 to 718-719 nm was observed. In the later stages of chloroplast differentiation, when xanthophylls and Chl b were synthesized, a higher-molecular-weight complex of PSI (CPIa) could be isolated. Pigment analysis demonstrated that CPIa contained xanthophylls and Chl b in addition to Chl a and ß-carotene. This indicates the formation of a light-harvesting antenna closely associated with PSI (LHCI). The addition of an LHCI to the reaction-center complex of PSI caused an increase in the absorption cross-section of PSI as shown by action spectroscopy and in-vivo fluorescence measurements. A model demonstrating the changes in the molecular organization of PSI during light-induced carotenoid biosynthesis in mutant C-6D of Scenedesmus obliquus is presented.

Evidence for an essential role of carotenoids in the assembly of an active photosystem II.

Dark-grown cells of mutant C-6D of the green alga Scenedesmus obliquus exhibit a high activity of photosystem I (PSI) but lack activity of photosystem II (PSII). These cells contain only the pigment-protein complex CPI, representing the reaction-center of PSI. Only chlorophyll a and precursors of carotenoids (lycopene, neurosporene, ξ-carotene, ß-zeacarotene) could be detected in dark-grown cells by analysis using high-performance liquid chromatography.Activity of PSII and the corresponding pigment-protein complex, CPa, develop immediately upon transfer to light. Light-harvesting complexes and higher molecular forms of PSI are synthesized only in the later stages of light-induced chloroplast differentiation. During illumination the amounts of carotenoid precursors decrease and carotenes, xanthophylls and chlorophylls a and b are formed. ß-Carotene and lutein are synthesized without a lag-phase. Their kinetics are similar to those of CPa formation and development of PSII activity. In contrast, all other xanthophylls are synthesized only after a lag-phase of about 30 min.Inhibition of the transformation of precursors into carotenoids by nicotine prevents the light-inducible development of PSII activity and CPa formation. During illumination under anaerobic conditions no xanthophylls are synthesized but high amounts of α- and ß-carotene accumulate. Such cells exhibit no PSII activity and show only traces of CPa. After subsequent transfer to aerobic conditions the xanthophylls are synthesized and simultaneously active PSII units are formed.The results prove that carotenoids are essential components for the assembly of active PSII units. Strong evidence is given that lutein is the absolute necessary prerequisite for this process. Whether ß-carotene is also an absolute necessary prerequisite for a functioning PSII unit cannot be deduced from our experiments.

Influence of energy flux and quality of light on the molecular organization of the photosynthetic apparatus in Scenedesmus.

The photosynthetic apparatus of the unicellular green alga Scenedesmus obliquus adapts to different levels and qualities of light as documented by the fluence-rate curves of photosynthetic oxygen evolution. Cultures adapted to low fluence rates of white light (5W·m(-2)) have more chlorophyll (Chl) per cell mass, a higher chlorophyll to carotenoid ratio and a doubling of the chlorophyll to cytochrome f ratio compared with cells adapted to high fluence rates of white light (20W·m(-2)). Only small differences can be observed in the halfrise time of fluorescence induction, the electrophoretic profile of the pigment-protein complexes and the Chl a/Chl b-ratio. Scenedesmus cells adapted to blue light of high spectral purity demonstrate, in comparison with those adapted to red light, a higher chlorophyll content, a higher ratio of chlorophyll to carotenoid and a much higher ratio of chlorophyll to cytochrome f. Regarding these parameters and the fluence-rate curves of photosynthesis, the blue light causes the same effects as low levels of white light. In contrast, the action of red light resembles rather that of high levels of white light. Blue-light-adapted Scenedesmus cells have a smaller Chl a to Chl b ratio, a faster half-rise time of fluorescence induction and more chlorophyll in the light-harvesting system than red-light-adapted cells, as shown in the electrophoretic profile of the pigment-protein complexes. Based on these results we propose a model for the adaptation of the photosynthetic apparatus of Scenedesmus to different levels and qualities of light. In this model low as compared with high levels of white light result in an increase in the number of photosystems per electron-transport chain, but not in an increase in the size of these photosystems. The same result is obtained by adaptation to blue light. The lack of sufficient Chl b formation in red-light-adapted cells results in a decrease in the light harvesting chlorophyll-protein complexes and a photosynthetic response like that found in cells adapted to high light levels. The findings reported here confirm our earlier results in comparing blue-and red-light adaptation of the photosynthetic apparatus with adaptation to low and high levels of white light, respectively.