Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.Abbreviations kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b-binding protein - M_r relative molecular mass - PSII photosystem II - SDS sodium dodecyl sulphate     

Observations on the Senescence of a Mutant Non-yellowing Genotype of Phaseolus vulgaris L.

A non-yellowing mutant of Phaseolus vulgaris L. was used toinvestigate factors involved in chlorophyll breakdown duringfoliar senescence. The mutant showed physiological changes similarto those of the normal yellowing type during senescence exceptthat leaf chlorophyll did not decline. Transmission electronmicroscope studies did not reveal appreciable differences inchloroplast ultrastructure between the two genotypes, suggestingthat chloroplast membrane integrity was not the factor preventingchlorophyll degradation in the mutant. However, the lack ofplastoglobuli in senescent mutant chloroplasts suggested thatthe lipid environment may be different from that of senescentnormal chloroplasts. Banding patterns of total soluble protein,resolved by sodium dodecyl sulphate-poly aery lamide gel electrophoresisshowed few, if any, differences between mature non-senescentnormal and mutant leaves; however, bands at 14 kD and 58 kDdiminished in senescent normal leaves, but remained in senescentmutant non-yellowing leaves. Key words: Non-yellowing mutant, Phaseolus vulgaris, senescence, chlorophyll degradation     

Leaf senescence in Festuca pratensis: accumulation of lipofuscinlike compounds

Fluorescent compounds (FCs) with spectral properties comparable to those of lipofuscin-like compounds are present in aqueous methanolic extracts of senescent meadow fescue, Festuca pratensis Huds., leaves. An HPLC system for the separation of FC from other fluorescent materials was developed. The chromatograms suggest that the FC-fraction consists of a large number of chemically related compounds. FCs are accumulated during senescence in leaves of a yellowing genotype, cv. Rossa. In leaves of a non-yellowing genotype, Bf 993, only traces of FCs appear at advanced stages of senescence.
FCs are regarded as final products of lipid peroxidation. Since both yellowing and non-yellowing genotypes are competent with regard to the degradation of galactolipids (the potential sources of polyunsaturated fatty acids) as well as regarding lipoxygenase (EC 1.13.11.12; a key enzyme of lipid peroxidation), and since incompentence to degrade chlorophyll is associated with lack of FC accumulation in the mutant genotype, it is hypothesized that the polar FCs present in senescent F. pratensis leaves represent catabolites of chlorophyll.     

Changes in chlorophyll and carotenoid contents in radish (Raphanus sativus) cotyledons show different time courses during senescence

Changes of chlorophylls and carotenoids from green to yellow cotyledons of the radish ( Raphanus sativus ) were simultaneously and systematically analysed by high-performance liquid chromatography (HPLC) with photodiode array detection. Twenty-one components, seven chlorophylls and 14 carotenoids, were detectable. Seven chlorophylls and five carotenoids were identified from the results of HPLC analyses. Most chlorophyll species degraded during senescence, whereas carotenoids showed different behaviour in their metabolism depending on pigment species. For instance, during senescence, the contents of lutein and violaxanthin changed only slightly, β-carotene in 5-day-senescent cotyledons became 2.7 times higher than non-senescent leaves. Carotenoids of radish cotyledons were classified into three groups by their changes in concentration during senescence (increased, degraded and constant) and their roles discussed.     

Lipid peroxidation in and photo-damage to a light-sensitive chlorescence pea mutant

Leaves of 10- to 12-day-old chlorescence lethal Pisum sativum L. mutant are similar to control plants with respect to the content of chlorophylls, carotenoids, fatty acids and α-tocopherol. Subsequent development of the mutant under high irradiation resulted in th destruction of the photosynthetic pigments, polyunsaturated fatty acids, α-tocopherol, and also in the accumulation of liposoluble fluorescent products. No increase in the level of malondialdehyde was observed. In chloroplasts isolated from mutant plants the contents of chlorophyll a and β-carotene were decreased to a greater extent than the more oxidized pigments (xanthophylls and chlorophyll b ). The data obtained are discussed with special reference to the role of lipid peroxidation in the injury of plant cells under the action of visible light and to the antioxidative mechanisms stabilizing photosynthetic membranes.     

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.     

The xanthophyll cycle is induced by light irrespective of water status in field-grown lavender (Lavandula stoechas) plants

The water relations, the photosynthetic capacity and the pigment content of leaves, i.e. chlorophylls, carotenes and xanthophylls, were analysed during the summer drought and recovery after autumn rainfalls in lavender ( Lavandula stoechas L.) plants grown in Mediterranean field conditions. Summer drought caused photoinhibition of photosynthesis and significant decreases in chlorophylls (by ca 75%), β -carotene (by ca 65%), and lutein and neoxanthin (by ca 50%), although their contents remained unaltered between predawn and midday, suggesting a progressive decrease in response to drought. In contrast, the levels of violaxanthin decreased from predawn to midday, giving rise to enhanced formation of zeaxanthin and antheraxanthin in high light. Zeaxanthin and antheraxanthin formation was not induced by water deficit. Although the levels of photosynthetic pigments were severely affected by water deficit, carotenoids decreased less than chlorophylls, which resulted in increased levels of carotenoids per unit of chlorophyll. We conclude that the enhanced formation of zeaxanthin in high light and the increased levels of carotenoids per unit of chlorophyll observed in water-stressed plants may help to avoid photoinhibitory damage to the photosynthetic apparatus.     

Changes in the chlorophylls and carotenoids of leaves of Nicotiana tabacum during senescence

In addition to chlorophylls a and b, β-carotene, lutein, violaxanthin and neoxanthin, leaves of tobacco (Nicotiana tabacum L. cv. Virginia Gold) contain antheraxanthin in some harvests. In lower leaves, chlorophylls decreased more rapidly than carotenoids during senescence, but both types of pigment decreased at equal rates in upper leaves. The chlorophyll a:b ratio decreased only in post-mature leaves. Total carotenoid decreased with age, with the relative proportion of β-carotene increasing in lower leaves. Seasonal influences rather than age of leaf determines whether antheraxanthin is present. No esterified xanthophylls were found in senescent leaves.     

Lipid and pigment composition of a chlorophyll b-deficient mutant of Chlamydomonas reinhardii

Lipids and pigments of the chlorophyll b -deficient mutant pg-113 and the parent strain (ps) of Chlamydomonas were analysed and compared. Monogalactosyldiglyceride, digalactosyldiglyceride, diacylglyceryl(N, N, N-trimethyl)homoserine, sulfoquinovosyldiglyceride, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol were found as major lipid components. While the lipid patterns were qualitatively and quantitatively almost the same in the two strains, the C₁₆/C₁₈ fatty acid ratios were different, 0.85 in the mutant and 1.11 in the parent strain. Furthermore, the relative amounts of C₁₆- and C₁₈-monoene fatty acids were slightly enhanced and the C₁₈-trienes slightly reduced in the mutant. In the parent strain, chlorophylls a and b , α- and β-carotene, lutein, violaxanthin, neoxanthin and loroxanthin were detected by HPLC. In the mutant, similar pigments were found, except that only traces of chlorophyll b and a reduced amount of neoxanthin were present. Since no chlorophyll-protein complex CP II could be detected in the mutant by electrophoresis, the possible interrelationships between pigment deficiency and alteration of chlorophyllprotein complexes are discussed.     

Eco-physiological studies on the response of taxodiaceous conifers to shading with special reference to the behavior of leaf pigments

The effects of light intensity on the content and composition of leaf pigments, especially of carotenoids, were studied with mature current-year leaves of Taxodiaceous saplings grown under different grades of shade in summer. Both chlorophyll and total carotenoid contents increased with decreasing light intensity, maintaining approximately linear relations between each other, over a range of relative solar radiation of 100% to 7% of full daylight. The regression of total chlorophyll content on mean solar radiation could be well approximated by Shinozaki-Kira's reciprocal equation. The ratio of chlorophyll a to chlorophyll b was smaller in the shade than in the sun. The percentage of α-carotene and violaxanthin in the total carotenoid content tended to increase with increasing degree of shade, while those of β-carotene and lutein were reduced. The eco-physiological meanings of the pigments were considered based on this evidence. The order of shade tolerance among the four species tested is also discussed taking the responses of leaf weight and chlorophyll content to incident light intensity into consideration.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis

The lipid compositions of leaves from Festuca pratensis cv. Rossa (yellowing) were compared with those from a non-yellowing mutant, Bf 993. The leaves of Bf 993 contained a higher level of acyl lipids on both a fresh-weight and a dry-weight basis. Diacylgalactosylglycerol, diacylgalabiosylglycerol and phosphatidylinositol were relatively enriched in the Bf 993 mutant while phosphatidylcholine was relatively reduced. There were no differences in the fatty-acid compositions of individual lipids between the two varieties. During senescence, the lipids of cv. Rossa were progressively degraded over an 8-d period. In contrast little lipid degradation was observed in the Bf 993 mutant during the first 4 d. The results support the hypothesis that the slower senescence changes of the Bf 993 mutant may be due, in part, to an altered membrane lipid composition.II=Thomas (1982b)     

Analysis of the chlorophyll catabolism pathway in leaves of an introgression senescence mutant of Lolium temulentum

Pigments, proteins and enzyme activity related to chlorophyll catabolism were analysed in senescing leaves of wild-type (WT) Lolium temulentum and compared with those of an introgression line carrying a mutant gene from stay-green (SG) Festuca pratensis. During senescence of WT leaves chlorophylls a and b were continuously catabolised to colourless products and no other derivatives were observed, whereas in SG leaves there was an accumulation of dephytylated and oxidised catabolites including chlorophyllide a, phaeophorbide a and 13(2) OH-chlorophyllide a. Dephytylated products were absent from SG leaf tissue senescing under a light-dark cycle. Retention of pigments in SG was accompanied by significant stabilisation of light harvesting chlorophyll-proteins compared with WT, but soluble proteins such as Rubisco were degraded during senescence at a similar rate in the two genotypes. The activity of phaeophorbide a oxygenase measured in SG tissue at 3d was less than 12% of that in WT tissue at the same time-point during senescence and of the same order as that in young pre-senescent WT leaves, indicating that the metabolic lesion in SG concerns a deficiency at the ring-opening step of the catabolic pathway. In senescent L. temulentum tissue two terminal chlorophyll catabolites were identified with chromatographic characteristics that suggest they may represent hitherto undescribed catabolite structures. These data are discussed in relation to current understanding of the genetic and metabolic control of chlorophyll catabolism in leaf senescence.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis

Soluble and thylakoid membrane polypeptides from senescing leaf tissue of Rossa, a normal yellowing Festuca pratensis genotype, were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and compared with those of the non-yellowing mutant Bf 993. Subunits of ribulose-1,5-bisphosphate carboxylase were the major soluble polypeptides and declined to low levels in senescing leaves of both genotypes. The major thylakoid polypeptides were those associated with the chlorophyllprotein complexes CPI and CPII. The levels of all thylakoid polypeptide species fell during senescence of Rossa leaf tissue but Bf993 lamellae retained CPI, CPII and a number of other hydrophobic low molecular weight polypeptides. The increasing hydrophobicity and decreasing protein complement of Bf 993 thylakoids were reflected in a fall in membrane density from 1.16 to 1.13 g cm^-3 over 8 d of senescence and a decline in the extractability of chlorophyll-containing membranes in the same period. In Bf993 the molar ratio of chlorophyll to hydrophobic membrane protein increased from 92 at day 0 to 296 at day 8. In the same time the ratio for Rossa increased from 88 to 722 and 8 d-senesced Rossa tissue yielded less than 2% of the solvent-soluble protein it contained at day 0 as compared with 24% for the protein of Bf993. These results are discussed in relation to the nature of the non-yellowing lesion.Abbreviations RuBPC ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - EDTA ethylenediaminetetraacetate - SDS sodium dodecyl sulphate - CP chlorophyll-protein complex     

Low growth temperature-induced changes to pigment composition and photosynthesis in Zea mays genotypes differing in chilling sensitivity

The effects on pigment composition and photosynthesis of low temperature during growth were examined in the third leaf of three chilling-tolerant and three chilling-sensitive genotypes of Zea mays L. The plants were grown under a controlled environment at 24 or 14 °C at a photon flux density (PFD) of 200 or 600 μ mol m^–2 s^–1. At 24 °C, the two classes of genotypes showed little differences in their photosynthetic activity and their composition of pigments. At 14 °C, photosynthetic activity was considerably reduced but the chilling-tolerant genotypes displayed higher photosynthetic rates than the chilling-sensitive ones. Plants grown at 14 °C showed a reduced chlorophyll (Chl) a + b content and a reduced Chl a / b ratio but an increased ratio of total carotenoids to Chl a + b . These changes in pigment composition in plants grown at low temperature were generally more pronounced in the chilling-sensitive genotypes than in the tolerant ones, particularly at high PFD. Furthermore, at 14 °C, all the genotypes showed increased ratios of lutein, neoxanthin and xanthophyll-cycle carotenoids to Chl a + b but a reduced ratio of β -carotene to Chl a + b , especially at high PFD. At 14 °C, the chilling-tolerant genotypes, when compared with the sensitive ones, were characterized by higher contents of β -carotene and neoxanthin, a lower content of xanthophyll-cycle carotenoids, a lower ratio of xanthophylls to β -carotene, and less of their xanthophyll-cycle carotenoid pool in the form of zeaxanthin. These differences between the two classes of genotypes were more pronounced at high PFD than at low PFD. The results are discussed in terms of the relationship that may exist in maize between pigment composition and the capacity to form an efficient photosynthetic apparatus at low growth temperature.     

On the role of ß-carotene in the reaction center chlorophyll a antennae of photosystem I

Absorption and low temperature fluorescence emission spectra were measured on chloroplast thylakoids and on purified reaction center chlorophyll a-protein complexes of photosystem I, CP-a₁. A clear association between the presence of ß-carotene and the occurrence of far red absorbing and emitting chlorophyll a components of the reaction center antennae of photosystem I was demonstrated. For this study chloroplasts and CP-a₁ were obtained from normal and carotenoid deficient plant material of various sources. The experimental material included 1) lyophilized pea chloroplasts extracted with petroleum ether, 2) the carotenoid deficient mutant C-6E of Scenedesmus obliquus and 3) wheat chloroplasts derived from normal and SAN-9789 treated plants. Removal of carotenoids, most likely principally ß-carotene, caused a loss of long wavelength absorbing chlorophylls in chloroplasts and purified CP-a₁, and the loss or diminution of the long wavelength peak seen in the low temperature fluorescence emission spectrum. This association between ß-carotene and special chlorophyll a forms may explain both the photoprotective and antenna functions ascribed to ß-carotene. In the absence of carotenoids in wheat and in the Scenedesmus mutant, the chlorophyll a antenna of photosystem I was extremely photosensitive. A triplet-triplet resonance energy transfer from chlorophyll a to ß-carotene and a singlet-singlet energy transfer from excited ß-carotene to chlorophyll would explain the photoprotective and antenna functions, respectively. The role of this association in determining some of the fluorescence properties of photosystem I is also discussed.     

Pigment ligation to proteins of the photosynthetic apparatus in higher plants

Ligation of pigments to proteins of the thylakoid membrane is a central step in the assembly of the photosynthetic apparatus in higher plants. Because of the potentially damaging photooxidative activity of chlorophylls, it is likely that between their biosynthesis and final assembly, chlorophylls will always be bound to protein complexes in which photooxidation is prevented by quenchers such as carotenoids. Such complexes may include chlorophyll carriers and/or membrane receptors involved in protein insertion into the membrane. Many if not all pigment-protein complexes of the thylakoid are stabilised towards protease attack by bound pigments. The major light-harvesting chlorophyll a/b protein (Lhebl,2) folds into its native structure in vitro only when it binds pigments. Pigment-induced folding may also be a general feature of chlorophyll-carotenoid proteins of the photosynthetic apparatus.     

Reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids

Reversed-phase high-performance liquid chromatography with octadecyl- or octylsilylated silica gel as the stationary phase provides a powerful tool in the analysis of chloroplast pigments from higher plants and green algae. Chromatographic columns packed with 10 μm chemically bonded silica gel particles allow the simultaneous separation of chlorophylls a and b, chlorophyll isomers, pheophytins a and b, α-carotene, β-carotene, lutein, violaxanthin, lutein-5,6-epoxide, antheraxanthin, neoxanthin and several minor carotenoids from a single sample within a short analysis time. The quantitative analysis requires a minimum of 1–5 pmol for carotenoids and 5–10 pmol for chlorophylls. Pigment degradation products, formed on polar stationary phases, are not found in reversed-phase high-performance liquid chromatography due to the weak hydrophobic forces on which the separation mechanism is based. The production of altered pigments however, either induced by various treatments or generated during the isolation, can be monitored as the reversed-phase system is selective enough to separate cis-isomers and oxidation products from their parent compounds. The reproducibility of the individual retention time for each pigment is better than ±1.5% which facilitates the identification of unknown pigments. The method is applied to the analysis of the pigment composition of Chlorella fusca, spinach (Spinacia oleracea) chloroplasts, and to the rapid determination of the ratio of chlorophyll a to chlorophyll b.     

Photosystem II efficiency in low chlorophyll,iron-deficient leaves

Iron deficiency (iron chlorosis) is the major nutritional stress affecting fruit tree crops in calcareous soils in the Mediterranean area. This work reviews the changes in PS II efficiency in iron-deficient leaves. The iron deficiency-induced leaf yellowing is due to decreases in the leaf concentrations of photosynthetic pigments, chlorophylls and carotenoids. However, carotenoids, and more specifically lutein and the xanthophylls of the V+A+Z (Violaxanthin+ Antheraxanthin+Zeaxanthin) cycle are less affected than chlorophylls. Therefore, iron-chlorotic leaves grown in either growth chambers or field conditions have increases in the molar ratios lutein/chlorophyll a and (V+A+Z)/chlorophyll a. These pigment changes are associated to changes in leaf absorptance and reflectance. In the chlorotic leaves the amount of light absorbed per unit chlorophyll increases. The low chlorophyll, iron-deficient leaves showed no sustained decreases in PS II efficiency, measured after dark adaptation, except when the deficiency was very severe. This occurred when plants were grown in growth chambers or in field conditions. However, iron-deficient leaves showed decreases in the actual PS II efficiency at steady-state photosynthesis, due to decreases in photochemical quenching and intrinsic PS II efficiency. Iron-chlorotic leaves were protected not only by the decrease in leaf absorptance, but also by down-regulation mechanisms enhancing non-photochemical quenching and thermal dissipation of the light absorbed by PS II within the antenna pigment bed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis Huds.

A study was made of linolenic acid-dependent oxidative chlorophyll bleaching (CHLOX) by thylakoid membranes from senescing leaf tissue of a normal cultivar (cv. Rossa) and a non-yellowing mutant genotype (Bf 993) of Festuca pratensis Huds. To overcome the problem of variation in levels of endogenous chlorophyll substrate in membranes from different sources, light-harvesting complex (LHC) was used to supplement thylakoid pigment. It was shown that CHLOX is associated with both Photosystem I and LHC-rich thylakoid subfractions but that purified LHC has negligible associated CHLOX activity and stimulates the rate of bleaching by isolated entire chloroplast membranes. Non-senescent tissue of Bf 993 and Rossa had essentially identical thylakoid CHLOX levels, which subsequently declined during senescence in darkness. The half-life of CHLOX from the mutant was three times greater than that of the normal genotype. In both cultivars, the amount of CHLOX assayed in thylakoids isolated at different times during senescence was more than adequate to support the corresponding in-vivo rate of pigment degradation as calculated from the half-life for chlorophyll. It was concluded that the non-yellowing mutation is not expressed through a lack of CHLOX activity. The role of linolenic acid metabolism in the regulation of thylakoid structure and function during senescence, and as a likely site of the non-yellowing lesion, are discussed.Abbreviations CHLOX linolenic acid-dependent oxidative chlorophyll bleaching activity - CHLPX chlorophyll peroxidase - CPI chlorophyll-protein complex I - LHC light-harvesting complex - LNA linolenic acid - PSI photosystem I - PSII photosystem II - S relative senescence rate - t _1/2 lialf time for degradation     

Effects of chlorophyll a, chlorophyll b, and xanthophylls on the in vitro assembly kinetics of the major light-harvesting chlorophyll a/b complex, LHCIIb

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem II in higher plants can be reconstituted with pigments in lipid-detergent micelles. The pigment-protein complexes formed are functional in that they perform efficient internal energy transfer from chlorophyll b to chlorophyll a. LHCIIb formation in vitro, can be monitored by the appearance of energy transfer from chlorophyll b to chlorophyll a in time-resolved fluorescence measurements. LHCIIb is found to form in two apparent kinetic steps with time constants of about 30 and 200 seconds. Here we report on the dependence of the LHCIIb formation kinetics on the composition of the pigment mixture used in the reconstitution. Both kinetic steps slow down when the concentration of either chlorophylls or carotenoids is reduced. This suggests that the slower 200 seconds formation of functional LHCIIb still includes binding of both chlorophylls and carotenoids. LHCIIb formation is accelerated when the chlorophylls in the reconstitution mixture consist predominantly of chlorophyll a although the complexes formed are thermally less stable than those reconstituted with a chlorophyll a:b ratio < or = 1. This indicates that although chlorophyll a binding is more dominant in the observed rate of LHCIIb formation, the occupation of (some) chlorophyll binding sites with chlorophyll b is essential for complex stability. The accelerating effect of various carotenoids (lutein, zeaxanthin, violaxanthin, neoxanthin) on LHCIIb formation correlates with their affinity to two lutein-specific binding sites. We conclude that the occupation of these two carotenoid binding sites but not of the third (neoxanthin-specific) binding site is an essential step in the assembly of LHCIIb in vitro.