Accumulation of the NON‐YELLOW COLORING 1 protein of the chlorophyll cycle requires chlorophyll b in Arabidopsis thaliana

Chlorophyll a and chlorophyll b are interconverted in the chlorophyll cycle. The initial step in the conversion of chlorophyll b to chlorophyll a is catalyzed by the chlorophyll b reductases NON‐YELLOW COLORING 1 (NYC1) and NYC1‐like (NOL), which convert chlorophyll b to 7‐hydroxymethyl chlorophyll a. This step is also the first stage in the degradation of the light‐harvesting chlorophyll a/b protein complex (LHC). In this study, we examined the effect of chlorophyll b on the level of NYC1. NYC1 mRNA and NYC1 protein were in low abundance in green leaves, but their levels increased in response to dark‐induced senescence. When the level of chlorophyll b was enhanced by the introduction of a truncated chlorophyllide a oxygenase gene and the leaves were incubated in the dark, the amount of NYC1 was greatly increased compared with that of the wild type; however, the amount of NYC1 mRNA was the same as in the wild type. In contrast, NYC1 did not accumulate in the mutant without chlorophyll b, even though the NYC1 mRNA level was high after incubation in the dark. Quantification of the LHC protein showed no strong correlation between the levels of NYC1 and LHC proteins. However, the level of chlorophyll fluorescence of the dark adapted plant (F_o) was closely related to the accumulation of NYC1, suggesting that the NYC1 level is related to the energetically uncoupled LHC. These results and previous reports on the degradation of chlorophyllide a oxygenase suggest that the a feedforward and feedback network is included in chlorophyll cycle.     

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.     

Phytol as one of the determinants of chlorophyll interactions in solution

Optical absorption and fluorescence parameters of chlorophyll a and the phytol-free chlorophyllide a, as well as of their Mg-depleted derivatives, were compared in a series of organic solvents. In contrast to prevailing opinion, the spectral properties of chlorophyll are not indifferent to the removal of phytol. The electronic absorption spectra of chlorophyll a and chlorophyllide a differ and display a different dependence on the nature of the solvent, which cannot be explained solely by the location of a charged carboxylic group in the proximity of the π– electron system. In fact, measurements in media of varying basicity show that deprotonation of the free carboxylic group in chlorophyllide, i.e., the presence of a negative point charge near the macrocycle, has no effect on pigment absorption spectra. Analysis of the solvent effect on the Q_Y energies in terms of solvent polarity reveals that the phytyl moiety perturbs the spectral features of chlorophyll, mainly due to its interactions with the pigment solvation shell. The phytyl residue might also be thus partly involved in controlling the central metal ligation in chlorophylls. This influence of phytol on the spectral features of chlorophyll should be taken into account when comparing the spectra in solution with various spectral forms of chlorophyll in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Chlorophyllide a is a metabolite late in the biosynthesis of chlorophylls and bacteriochlorophylls. Isolation procedures for chlorophyllide a from Rhodobacter capsulatus CB1200 and barley (Hordeum vulgare L.) are described and compared. R. capsulatus CB1200 is a double mutant in the bacteriochlorophyllide a biosynthetic pathway, and chlorophyllide a is excreted by the cells when grown in Tween 80-containing liquid medium. It was purified by liquid or solid phase extraction, yielding 7 mg of chlorophyllide a from 1 L of culture. In a second approach, intrinsic chlorophyllase activity was used to dephytylate chlorophyll in an acetonic preparation of leaves of wild-type or chlorophyll b-deficient barley. Purification was achieved by liquid phase extraction, yielding 14 μg of chlorophyllide a per gram of barley leaves. Chlorophyllide a was identified by thin layer chromatography, absorption spectroscopy, and mass spectrometry.     

Biosynthesis of chlorophyll b in pigment mutant C-2A' of Scenedesmus obliquus

It is demonstrated that chlorophyll b does not only derive from chlorophyll a , but is also formed separately from an in vivo-occurring chlorophyllide b . The branching point for the latter synthesis is at the level of chlorophyllide, since no protochlorophyllide b was detectable. We have indications that the enzyme oxidizing chlorophyll a to chlorophyll b accepts also non-phytylated 17,18 dihydroporphyrins and is not restricted to chlorophylls. Preparations of chlorophyllide a and chlorophyll a could both be transferred with the same enzyme fraction to chlorophyllide b and chlorophyll b , respectively. Preliminary experiments show this enzyme to be membrane bound and light independent. An updated scheme for chlorophyll b biosynthesis is presented.     

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.     

Esterification and Spectral Shifts of Chlorophyll(ide) in Wildtype and Mutant Seedlings Developed in Darkness

Spectral changes and esterification (presumably with phytol) of newly formed chlorophyllide a in dark-grown leaves of wildtype bean (Phaseolus vulgaris) and barley (Hordeum vulgare) and a number of chloroplast mutants in barley, were studied by spectrofluorimetry on leaves and on solvent extracts. The shift of the fluorescence emission maximum from 692–694 to 678 nm (excitation shift: 682–684 to 672 nm) and esterification of chlorophyllide a have a similar time course, and both processes are temperature dependent in a similar manner. After completion of the spectral shift and esterification, the fluorescence efficiency of chlorophyll a increases with a subsequent reaccumulation of protochlorophyllide. In leaves of mutants where the shift of fluorescence from 692 to 678 nm is lacking, esterification and the subsequent processes are also blocked. In leaves of mutants with a rapid shift of the fluorescence from 692 to 678 nm, or with direct photoconversion to chlorophyllide a with the fluorescence at 678 nm, esterification is also rapid. The results are interpreted as a sequence of molecular events involving a conformational relaxation of the chlorophyllide holochrome and a translocation of chlorophyll a to reaction centers of the photosystems.     

Control of chlorophyll synthesis by phytochrome

Summary The rate of chlorophyllide esterification in mustard cotyledons can be increased by a pretreatment with 5 min red light applied 24 h prior to the protochlorophyll(ide)chlorophyll(ide) photoconversion at 60 h after sowing. Simultaneously the red light pulse pretreatment leads to a decrease of the total amount of chlorophyll(ide) a in darkness. It has been proven that phytochrome (P_fr) is the photoeffector for both. Since the amounts of esterified chlorophyllide are determined by the ratio [chlorophyll a]/[chlorophyllide a+chlorophyll a] it is assumed that P_fr increases the rate of esterification indirectly via stimulating the decrease of chlorophyll(ide) a. The regulation of chlorophyll synthesis by P_fr does not seem to involve a control of esterification. The duration of the chlorophyllide esterification differs from the duration of the Shibata shift although both are greatly shortened by the red light pulse pretreatment. The effect of 5 min red light on the duration of the esterification is fully reversible by 5 min far-red light while the reversibility with respect to the Shibata shift is lost within 2 min [Jabben, M. and H. Mohr, Photochem. Photobiol. 22, 55–58 (1975)]. We conclude that the control of the chlorophyllide esterification and the control of the Shibata shift cannot be traced back to the same initial action of P_fr.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Chl(ide) sum of Chl and Chlide - PChl protochlorophyll - PChlide protochlorophyllide - PChl(ide) sum of PChl and PChlide - P_fr far-red absorbing form of the phytochrome system     

Albinism in plants – far beyond the loss of chlorophyll: Structural and physiological aspects of wild‐type and albino royal poinciana (Delonix regia) seedlings

• The partial or complete loss of chlorophylls, or albinism, is a rare phenomenon in plants. In the present study, we provide the first report of the occurrence in albino Delonix regia seedlings and describe the morpho‐physiological changes associated with albinism.
• Wild‐type (WT) and albino seedlings were characterized. Leaflets samples were processed following common procedures for analysis with light, scanning and transmission electron microscopy. The chlorophyll a fluorescence parameters and the carbohydrate, lipid and soluble protein content were also determined in leaf and cotyledon samples of both albino and WT seedlings.
• Albino seedlings showed reduced growth. They also had lower chlorophyll and protein content in foliar tissues than WT seedlings, in addition to lower concentrations of lipids and carbohydrates stored in cotyledons. The chloroplasts of albino seedlings were poorly developed, with an undefined internal membrane system and the presence of plastoglobules. Wild‐type seedlings had a uniseriate and hypoestomatic epidermis. The mesophyll was dorsiventral, consisting of a layer of palisade parenchyma and two to four layers of spongy parenchyma. In albino seedlings, the spongy parenchyma was compact, with few intercellular spaces, and the thickness of the mesophyll was larger, resulting in increased thickness of the leaf blade. Albino seedlings had higher stomatal density and number of pavement cells, although the stomata had smaller dimensions.
• In addition to the partial loss of chlorophylls, albino D. regia showed changes at physiological and structural levels, demonstrating the crucial nature of photosynthetic pigments during the development and differentiation of plant leaf tissues/cells.

     

Peroxidase-catalysed oxidation of chlorophyll by hydrogen peroxide

Chlorophyll is effectively bleached by H₂O₂ in the presence of certain phenols and peroxidase (EC 1.11.1.7) extracted from acetone powders of orange flavedo (Citrus sinensis). Optimal conditions for chlorophyll: hydrogen peroxide oxidoreductase include: pH, 5.9; [H₂O₂] 222 μM; ionic strength 0.11. A phenol is required and resorcinol is the most effective. Catechol and hydroquinone are inhibitory. Chlorophyll a, chlorophyllide a, and chlorophyll b all have similar V_max but K_m for chlorophyll a is about one-third that of chlorophyll b, while the K_m for chlorophyllide a is about one-half that of chlorophyll a. Pheophytin a was much less reactive than chlorophyll a, and Mg²⁺ included in the reaction system did not affect rates of pheophytin destruction.     

Studies on chlorophyll biosynthesis and other things

Our research on chlorophyll biosynthesis, over a period of approximately twenty years, has been described, emphasizing those areas in which our laboratory made significant and timely contributions. References to some of our most important articles are included. Portions of the chlorophyll biosynthetic pathway, in which our own laboratory was not involved, for example, the reduction of protochlorophyllide to chlorophyllide and the phytylation of the latter to yield chlorophyll a, have not been covered in this article. Those events which preceded my involvement with chlorophyll biosynthesis, but which contributed to the formation of my own scientific personality, are mentioned briefly in the Introduction. My non-scientific avocations have been included at the request of the reviewers and Govindjee.     

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.     

NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll – protein complexes during leaf senescence

Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll – protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4‐1, a stay‐green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4‐1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation‐related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity‐dependent manner. Surprisingly, the F_v/F_m value remained high in nyc4‐1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4‐1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild‐type and other non‐functional stay‐green mutants, including sgr‐2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll – protein complexes during leaf senescence is discussed.     

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.     

Interconversions of Chlorophylls a and b Synthesized from Exogenous Chlorophyllides a and b in Etiolated and Post-Etiolated Rye Seedlings

A comparative study of reciprocal conversions of chlorophylls a and b (Chl aand Chl b) in etiolated and post-etiolated rye seedlings (Secale cereale L.) was performed. The production of these pigments was initiated by infiltration of exogenous chlorophyllides a and b (Chlide a and b). It was shown that Chlide b, when infiltrated into etiolated rye seedlings, was esterified, producing Chl b. A major portion of Chl b (more than 80%) was transformed into Chl aduring long-term seedling dark exposure. The high rate of Chl b conversion into Chl a in the pool of pigments of exogenous origin was also observed during the lag-phase when there was no chlorophyll formation from endogenous precursors. The infiltration of Chlide a resulted in Chl a formation. The efficiency of its conversion into Chl b was low (about 1%) in the etiolated seedlings but increased during their greening. In the post-etiolated seedlings infiltrated with Chlide b, which were preliminary illuminated for 6–12 h, the Chl /Chl a ratio was almost similar in the pools of pigments synthesized from both exogenous and endogenous precursors. The rates of direct and reverse reactions responsible for the interconversion of Chl aand Chl b depended on the stage of the formation of the photosynthetic apparatus during greening of etiolated seedlings, when the particular structural components are formed in a definite sequence.     

The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in chlorophyll (Chl) synthesis, and is required for prolamellar body (PLB) formation in etioplasts. Rice faded green leaf (fgl) mutants develop yellow/white leaf variegation and necrotic lesions during leaf elongation in field‐grown plants. Map‐based cloning revealed that FGL encodes OsPORB, one of two rice POR isoforms. In fgl, etiolated seedlings contained smaller PLBs in etioplasts, and lower levels of total and photoactive Pchlide. Under constant or high light (HL) conditions, newly emerging green leaves rapidly turned yellow and formed lesions. Increased levels of non‐photoactive Pchlide, which acts as a photosensitizer, may cause reactive oxygen accumulation and lesion formation. OsPORA expression is repressed by light and OsPORB expression is regulated in a circadian rhythm in short‐day conditions. OsPORA was expressed at high levels in developing leaves and decreased dramatically in fully mature leaves, whereas OsPORB expression was relatively constant throughout leaf development, similar to expression patterns of AtPORA and AtPORB in Arabidopsis. However, OsPORB expression is rapidly upregulated by HL treatment, similar to the fluence rate‐dependent regulation of AtPORC. This suggests that OsPORB function is equivalent to both AtPORB and AtPORC functions. Our results demonstrate that OsPORB is essential for maintaining light‐dependent Chl synthesis throughout leaf development, especially under HL conditions, whereas OsPORA mainly functions in the early stages of leaf development. Developmentally and physiologically distinct roles of monocot OsPORs are discussed by comparing with those of dicot AtPORs.     

The Pool of Chlorophyllous Pigments in Barley Seedlings of Different Ages under Heat Shock and Water Deficit

The effects of a high temperature (3 h, 40°C) and water deficit (45 h on 3% PEG 6000) on the pool of chlorophyllous pigments in the leaves of 4-, 7-, and 11-day-old barley (Hordeum vulgare L.) seedlings were studied. Heating resulted in a decrease in the total content of chlorophylls (Chl) (a + b) in 4-day-old plants but not in the older leaves. Water deficit induced an increase in the pigment content in young seedlings but reduced it in the leaves of 11-day-old plants. In young seedlings, hyperthermia and dehydration affected similarly Chl (a + b) degradation, leading to a marked inhibition of the chlorophyllase (Chlase) activity hydrolyzing Chl to chlorophyllides and phytol. In old leaves, an activation of this enzyme was observed. The stress factors under study affected different stages of pigment biosynthesis. High temperature inhibited the activity of dark and light stages of Chl(a + b) biosynthesis. Dehydration did not change markedly the resynthesis of protochlorophyllide, while the enzymes of the light stage of Chl biosynthesis were activated in young but inhibited in old barley leaves. The results thus obtained allowed us to conclude that heat treatment and dehydration specifically affected the Chl biosynthesis. At the same time, the Chlase response was nonspecific.