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.     

The turnover of chlorophyll in greening wheat leaves

A method for the estimation of chlorophyll turnover in wheat leaves is presented. This is based on the inhibition of chlorophyll synthesis by treatment of the cut leaves with laevulinic acid (LA), a competitive inhibitor of δ-aminolaevulinic acid dehydratase. The turnover of chlorophyll in young, greening leaves, given short periods of light was a relatively rapid process. However, in seedlings exposed to light for longer periods the turnover became progressively slower, and was measured in days rather than hours.     

Degradation of proteins from thylakoid membranes in senescing wheat leaves at high temperature

This investigation determined whether thylakoid proteins would be degraded more rapidly or not in senescing wheat (Triticum aestivum L. em. Thell.) leaves concurrently exposed to high temperatures. Excised leaves were pulse-labelled with [³⁵S]-methionine for a 12 h period, and then incubated at 22,32 or 42°C for 0, 1, 2, or 3 d, before extracting a thylakoid enriched membrane sample. After electrophoretic separation, two prominent [³⁵S]-labelled protein bands were chosen for further analyses. Band A contained the D-1 thylakoid protein and band B contained thylakoid proteins of the light harvesting complex (LHCII) associated with photosystem II (PSII). Total protein, [³⁵S]-labelled protein, band A protein, and band B protein within the thylakoid enriched membrane samples were measured. Unlabelled thylakoid enriched membrane samples, extracted from leaves given similar treatments, were used to measure uncoupled whole-chain and photosystem II (PSII) electron transport and chlorophyll fluorescence. Accentuated decline in whole-chain and PSII electron transport, increasing F_o values, and decreasing F_max values were a result of high temperature injury in leaves treated at 42°C. None of the thylakoid enriched membrane protein fractions were degraded more rapidly in high-temperature treated leaves. Degradation of the total [³⁵S]-labelled membrane proteins and band B was inhibited by the 42°C treatment. The results indicate that high temperature stress may disrupt some aspects of normal senescence.     

Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments

Total peroxidase activity increased during senescence of excised barley ( Hordeum vulgare L. cv. Kashimamugi) leaves. Kinetin treatment furter increased total peroxidase activity but repressed chlorophyll degradation in excised barley leaves. When isoperoxidases were extracted from barley leaf segments. 4 cationic and 4 anionic isozymes were found in polyacrylamide gel electrophorests during leaf senescence. The chloroplasts contained only two cationic isoperoxidase activities. One (designated C4) was repressed by kinetin. and the other (C3) was increased by kinetin. Glucosamine, which also repressed the degradation of chlorophyll, completely repressed C4 activity but did not affect C3 activity. The induction with senescence, and the repression with kinetin and glucosamine, suggest chat chloroplast isoperoxidase C4 may function as a chlorophyll-degrading enzyme during barley leaf senescence.     

Isoenzymes of naphthyl acetate esterase in senescing leaves of Festuca pratensis

Naphthyl acetate esterase (NAE) of leaves of Festuca pratensis had an apparent MW of 55000. Five major NAE isoenzymes were resolved by gel electrophoresis. During leaf senescence the proportions of these isoenzymes altered and two novel isoenzymes became active. Cycloheximide applied to leaves delayed and diminished the responses of NAE isoenzymes during senescence. The two novel NAEs were similar in MW and substrate affinity to pre-existing NAEs. Partially-purified NAE had no cholinesterase, carboxypeptidase, ethyl acetate esterase or ethyl butyrate esterase activity. Lack of inhibition by eserine, PCMB and organophosphorus insecticide classified these enzymes as acetylesterases.     

Influence of Cd2+ on growth, chlorophyll content, and water relations in young barley plants

Barley (Hordeum vulgare L., cv. Hemus) plants were grown in nutrient solution with or without 54 μM Cd²⁺ for 12 d. A treatment with Cd²⁺ inhibited the growth of young barley plants. The main factor limiting plant growth was net assimilation rate, due to decreased photosynthetic rate and accelerated dark respiration rate. One of the reasons for the reduced photosynthetic rate was the lower chlorophyll and carotenoid content. Cd²⁺ decreased water potential and transpiration rate, but relative water content in leaves of the treated plants was not significantly changed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of kinetin on chlorophyll synthesis in ageing etiolated barley leaves exposed to light

Etiolated barley seedlings lose the ability to produce chlorophyll and soluble protein on exposure to light with increasing age. Similarly, the production of δ-aminolaevulinic acid-dehydratase and succinyl-CoA synthetase is decreased in older etiolated leaves exposed to light. The rate of protochlorophyllide₆₅₂ regeneration decreased well before the rates of exogenous δ-aminolaevulinic acid conversion to protochlorophyllide₆₃₂ was affected by ageing. Application of kinetin retarded these ageing symptoms in the etiolated leaves.     

The effect of haem on chlorophyll synthesis in barley leaves

Exogenously supplied bovine haemin, fed to etiolated barley leaves, inhibited chlorophyll synthesis in leaves exposed to light. Haemin inhibited the regeneration of protochlorophyllide (P650) and the conversion of exogenously supplied δ-aminolaevulinate (ALA) to protochlorophyll (P630). The effect of haemin on chlorophyll production was overcome by incubating the leaves in water in the dark before light treatment, suggesting the operation of a rapid haem destruction mechanism in leaves. Protohaem turnover in dark-grown leaves was between 8 and 9 hr, based on the rate of degradation of erogenous haemin and the rate of protohaem breakdown in laevulinic acid (LA) treated leaves. The rate constant for haem destruction was 85 pmol/nmol/hr in the dark and 45 pmol/nmol/hr after 4 hr light. There was no evidence that light affects the synthesis of protohaem. It appears that the regulation of endogenous levels of protohaem is by breakdown and it is this mechanism which is under light control. Haem considerably decreased the incorporation of radioactivity from glycollate-[¹⁴C], glycine-[¹⁴C] and glutamate-[¹⁴C] into accumulated ALA in the presence of LA.     

Aromatic aldehyde constituents of graminaceous cell walls

p-Hydroxybenzaldehyde, vanillin and syringaldehyde were released as their sodium salts from graminaceous cell walls by treatment with sodium hydroxide. Treatment of the walls with ‘cellulase’ having both cellulase and hemicellulase activity released the aldehydes in bound form apparently linked at their phenolic groups to the wall polysaccharides. These findings are discussed in relation to tests for lignin using phloroglucinol-HC1 and alkaline nitrobenzene reagents.     

Possible mechanisms of light-induced chlorophyll degradation in senescing leaves of Hydrilla verticillata

Light treatment markedly accelerated the chlorophyll loss in senescing leaves of Hydrilla verticillata [(L.f.) Royle] as compared to dark treatment, whereas such acceleration could not be observed in senescing spinach (Spinacia oleracea L.) leaves. The light-induced cholorophyll loss in Hydrilla was retarded slightly by chloramphenicol and markedly by cycloheximide. Catalase (EC 1.11.1.6) activity did not change appreciably in Hydrilla leaves either in light or in darkness, while in spinach it declined markedly in the dark, and light retarded such decline. Peroxidase activity in Hydrilla showed faster increase in light than in darkness, while in spinach it increased only in light during senescence. The activity of phenol(pyrogallol)-specific peroxidase increased markedly in light, and that of ascorbate-specific peroxidase decreased slightly both in light and darkness during senescence of Hydrilla leaves. This rise in phenolspecific peroxidase activity was prevented by cycloheximide treatment. Pretreatment of Hydrilla leaves with monophenol (2,4-dichlorophenol) and o-diphenol (hydroquinone) accelerated and retarded, respectively, the light-induced cholorophyll loss. Pretreatment of Hydrilla leaves with H₂O₂ augmented the chlorophyll loss more markedly in light than in darkness. The endogenous level of H₂O₂ increased more in light than in dark during senescence of Hydrilla leaves. Treatment of Hydrilla leaves with 3-(3.4-dichlorophenyl)-l,l-dimethylurea. a photosystem II inhibitor, prevented both light-induced rise in H₂O: level and chlorophyll loss, but it was without effect in the dark. Retardation of light-induced chlorophyll loss occurred during senescence of Hydrilla leaves when light was given in different photoperiods in a 24-h daily cycle for 6 days instead of as continuous irradiance. There was a negative correlation between the length of the photoperiod and the extent of cholorophyll loss.     

Modulation of leaf elongation, tiller appearance and tiller senescence in spring barley by far-red light

Supplemental far-red (FR) illumination of light-grown grass seedlings inhibits tiller production while enhancing leaf elongation. Although much is known about FR enhancement of internode elongation in dicots, relatively little research has been conducted to determine the effects of FR on monocot development. In growth chamber experiments, fibre optics were used to direct supplemental FR to elongating leaf blades, main stem bases and mature leaf blades of light-grown barley (Hordeum vulgare L.) seedlings. Our objective was to identify specific sites of perception for FR enhancement of leaf elongation and inhibition of tiller production, and to assess potential FR effects on tiller senescence. Far-red illumination of elongating leaves or of the main stem base reduced the total number of tillers per plant, primarily by reducing secondary and tertiary tiller production, and enhanced leaf elongation. However, leaf elongation was less sensitive to stem base treatments than to illumination of the elongating blade. Increased leaf length resulted from increased leaf elongation rate, while the duration of leaf elongation was unaffected. Exposure of mature leaf blades to FR had no effect on tillering or leaf elongation. None of the FR treatments led to tiller senescence. Localization of FR perception in vertically oriented tissues such as elongating blades and stem bases permits early detection of reflected light from neighbouring plants, allowing rapid response to impending competition.     

Methionine metabolism and ethylene biosynthesis in senescing petals of Tradescantia

The endogenous content of methionine in isolated petals of Tradescantia was found to increase during petal senescence while the levels of S-methylmethionine and protein were found to decline. The increase in free methionine was, at least in part, the result of protein degradation. Methionine and homocysteine were shown to be intermediates in ethylene biosynthesis while S-methylmethionine was not involved. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to all floral tissues resulted in large stimulations of ethylene production. ACC was shown to be an endogenous amino acid the internal levels of which correlated positively with the rate of ethylene production. Application of l-methionine-[U-¹⁴C] led to a rapid appearance of radioactivity in both ethylene and ACC. The specific radioactivity of C-2 and C-3 of ACC and that of ethylene were found to be nearly identical which indicated that ACC was the immediate precursor of ethylene in senescing petals of Tradescantia.     

Haem and chlorophyll formation in etiolated and greening leaves of barley

The amounts of protochlorophyllide (P650) and protohaem were measured in ageing dark-grown barley leaves. Maximum amounts of P650 and protohaem were found in 6- to 8-day-old material after which P650 declined rapidly and protohaem more slowly. In leaves exposed to light maximum chlorophyll was produced in 6-day-old material with progressively less the older the leaves. Haem concentrations increased in seedlings of all ages exposed to light. A lag phase was observed for both chlorophyll and haem formation in leaves given a light treatment. Haem, however, showed a slight yet sig nificant decline as chlorophyll production commenced. The results indicate that chlorophyll and haem synthesis share a common pool of δ-aminolae vulinic acid (ALA). At a certain stage of development, the magnesium porphyrin pathway diverts precursors away from haem synthesis. It is only when the ALA synthesising system is well developed that the production of ALA can satisfy pathways to both haem and chlorophyll. The observed changes in haem under certain conditions suggest that, as in animal systems, haem levels may regulate porphyrin formation (chlorophylls) by controlling the supply of ALA.     

Glycine metabolism and chlorophyll synthesis in barley leaves

α-Hydroxypyridine methane sulphonic acid (HPMS), isonicotinyl hydrazide (INH) and nialamide inhibit chlorophyll synthesis in etiolated barley leaves exposed to light. HPMS lowered the rate of protochlorophyllide regeneration but had little effect on the synthesis of protochlorophyll (P630) from exogenous $\text{δ}$-aminolaevulinic acid (ALA). The addition of glycine to HPMS treated leaves partially overcame the inhibition of chlorophyll synthesis. Glycine-[¹⁴C] was readily incorporated into ALA in dark-grown leaves. HPMS treatment increased the sp. act. of ALA in leaves fed glycine-[¹⁴C]. Glycollate oxidation was lower in extracts from HPMS treated leaves. Plants may therefore have two pathways for ALA production with the glutamate pathway becoming more important in conditions where photorespiration is high.     

Ultrastructural changes in aging leaves of a light-grown achlorophyllous mutant of barley

The pattern and sequence of cellular degradation during the course of leaf senescence remains obscure and the nature of the trigger that induces cell senescence is unknown. In order to probe the pre-mortem phase of senescence temporal changes in cell ultrastucture were studied in aging leaves of light-grown achlorophyllous Hordeum vulgare L. cv. Dyan mutant seedlings. Electron microscope examination of the ultrastructure of mesophyll cell plastids revealed the absence of ribosomes and a highly disorganized prolamellar body. Both the number and size of plastoglobuli increased with aging and this change coincided with depletion of starch grains and dilation of lamellar membranes. Aging of mesophyll cells occurred coincident with a decline in ribosome content of the cytoplasm and loss of matrix granularity. Loss of ribosomes associated with the outer nuclear envelope membrane and a reduction in chromatin were also apparent. Only after 10 days was there evidence of loss of internal membrane integrity and swelling of mitochondrial cristae. Compartmentation was thus maintained during the aging process with membrane dissolution occurring late in senescence. These results suggest that an inability to produce chlorophyll and carotenoids and form thylakoid stacks due to the absence of plastid ribosomes, contributes to the rapid onset of senescence in light-grown achlorophyllous seedlings. Furthermore, disruption of chloroplast ribosome synthesis/assembly may constitute part of the plastid signal involved in triggering cell senescence.     

Light and electron microscope studies on pollen development in barley (Hordeum vulgare L.) grown under copper-sufficient and deficient conditions

Abstract. Pollen development in copper-deficient barley plants is highly irregular resulting in low and variable pollen fertility. The main cause of this sterility was found to be the abnormal development of the tapetum which becomes expansionary and invasive as the pollen develops. The ultrastructure of both tapetum and microspores is different from that of control material with irregularities of exine deposition, endopolyploidy of tapetal nuclei and an alteration of organelle composition being correlated with low fertility.     

Effect of carnitine on greening barley leaves

Carnitine increases chlorophyll production in greening barley leaves. [Methyl-¹⁴C]carnitine fed to greening leaves was not utilized as a carbon sou     

Compartmentation of phenylacetic and cinnamic acid synthesis in spinach

Applying labelled phenylalanine or tyrosine to purified intact spinach chloroplasts, only the corresponding phenylacetic acids but not the cinnamic acids could be detected. The addition of mercaptoethanol or dl -dithiothreitol and the variation of light conditions had only a slight effect. However, cinnamic acids could be found together with phenylacetic acids in leaf homogenates indicating the presence of phenylalanine and/or tyrosine ammonia lyase outside the spinach chloroplasts. Similar results were obtained with barley leaf homogenates, where cinnamic acids were the main products. Reviewing recent findings on amino acid synthesis in spinach leaves, it may be concluded that the synthesis of aromatic amino acids is restricted to the chloroplast, whereas the metabolism of secondary aromatic compounds is predominantly localized outside the chloroplasts.