Pheophytin Pheophorbide Hydrolase (Pheophytinase) Is Involved in Chlorophyll Breakdown during Leaf Senescence in Arabidopsis

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll → pheophytin → pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.     

Detection of Chlorophyll Breakdown Products in the Senescent Leaves of Higher Plants

The determination of intermediary breakdown products of chlorophyllsin senescent leaves of higher plants was performed by high-performanceliquid chromatographic technique that had been newly developedto separate and identify the oxidation products of chlorophylls,in particular monopyrrole derivatives [Suzuki et al. (1999)J. Chromatogr. A 839: 85]. In six plants tested, degradationproducts were detected only in the senescent leaves of plantsincluding barley (Hordeum vulgare), except for radish (Raphanussativus), in which they were found both in pre- and senescentcotyledons. In the senescent cotyledons of barley, three degradationproducts, hematinic acid, methyl ethyl maleimide, and the putativedegraded C-E-ring derivative, methyl vinyl maleimide dialdehyde,were detected. In addition to above three products, methyl vinylmaleimide was also found in both pre- and senescent cotyledonsof radish. These products decreased during senescence with anaccumulation of unknown compound(s), probably degraded monopyrrolederivatives. The degradation process and amounts of breakdownproducts of chlorophylls depend largely on plant species andvary with length of senescence. To conclude, it is likely thatchlorophylls are degraded into low-molecular-weight hydrolyticcompounds through monopyrroles. (Received March 10, 1999; Accepted June 11, 1999)     

Involvement of a Putative Bipartite Transit Peptide in Targeting Rice Pheophorbide a Oxygenase into Chloroplasts for Chlorophyll Degradation during Leaf Senescence

Leaf senescence is one of the major factors contributing to the productivity and the grain quality in crops. The regulatory mechanism of leaf senescence remains largely unknown. Here, we report the identification and characterization of a rice early senescence 1(eas1)mutant, which displayed an early leaf senescence phenotype, accompanying by dwarfism and reduced tiller number, eventually leading to the reduction of grain yield. Map-based cloning revealed that the nuclear gene EAS1 encodes a pheophorbide a oxygenase(PaO), a key enzyme for chlorophyll breakdown. A highly conserved Thr residue of PaO was mutated into Ile in the eas1 mutant. Phylogenetic analysis indicates that PaO is an evolutionarily conserved protein, and EAS1 is 68% identical to the Arabidopsis ACCERLERATED CELL DEATH(ACD1) protein. Unlike ACD1 that contains a single transit peptide, EAS1 contains two putative transit peptides at its N-terminus, which are essential for its functionality, suggesting that targeting of EAS1 to the chloroplast is likely mediated by a putative bipartite transit peptide. Consistently, only a short version of EAS1 lacking the first putative transit peptide, but not the full-length EAS1,was capable of rescuing the Arabidopsis acd1 mutant phenotype. These results suggest that rice EAS1 represents a functional PaO, which is involved in chlorophyll degradation and may utilize a unique mechanism for its import into the chloroplast.     

Chlorophyll Catabolites in Senescent Leaves of the Lime Tree (Tilia cordata)

In cold extracts of senescent leaves of the Lime tree (Tilia cordata), two colorless nonfluorescent chlorophyll catabolites (NCCs) were identified, named Tc‐NCC‐1 and Tc‐NCC‐2, as well as a polar yellow chlorophyll catabolite (YCC), named Tc‐YCC. The constitution of the two NCCs was determined by spectroscopic means. In addition, a tentative structure was derived for Tc‐YCC. The three chlorophyll degradation products exhibited tetrapyrrolic structures, as are typical of NCCs or YCCs, and turned out to be rather polar, due to a glucopyranosyl group at their 8²‐position. At their 3‐positions, the more polar Tc‐NCC‐1 carried a 1,2‐dihydroxyethyl group and the less polar Tc‐NCC‐2 a vinyl group. Tc‐YCC was identified as the product of an oxidation of Tc‐NCC‐1.     

Chlorophyll Catabolites in Senescent Leaves of the Plum Tree (Prunus domestica)

In cold extracts of senescent leaves of the plum tree (Prunus domestica ssp. domestica), six colorless non‐fluorescent chlorophyll catabolites (NCCs) were characterized, named Pd‐NCCs. In addition, several minor NCC fractions were tentatively classified. The structure of the most polar one of the NCCs, named Pd‐NCC‐32, featured an unprecedented twofold glycosidation pattern. Three of the NCCs are also functionalized at their 3²‐position by a glucopyranosyl group. In addition, two of these glycosidated NCCs carry a dihydroxyethyl group at their 18‐position. In the polar Pd‐NCC‐32, the latter group is further glycosidated at the terminal 18²‐position. Four other major Pd‐NCCs and one minor Pd‐NCC were identified with five NCCs from higher plants known to belong to the ‘epi’‐series. In addition, tentative structures were derived for two minor fractions, classified as yellow chlorophyll catabolites, which represented (formal) oxidation products of two of the observed Pd‐NCCs. The chlorophyll catabolites in leaves of plum feature the same basic structural pattern as those found in leaves of apple and pear trees.     

Breakdown of Galactolipids in Senescent Barly Leaves

The changes of galactolipids (MGDG and DGDG, largely 18:3/18:3), free fatty acids (FFA), and phosphatidylcholine (PC) taking place during senescence of primary barley leaves were analysed employing HPLC and GLC. Upon induction of senescence MGDG and, with some delay, DGDG began to disappear and were largely broken down at the end of the senescence period. A concomitant appearance of a pool of FFA could not be observed. However, PC accumulated during the main period of galactolipid breakdown. This change was due to the marked increase of the 18:3/18:3 molecular species of PC. An inverse correlation between the changes of galactolipids and PC could be established. A hypothesis featuring the conversion of galactolipids via diacylglycerol to PC is presented as the principal route of galactolipid breakdown.     

Conversion of Chlorophyll b to Chlorophyll a and the Assembly of Chlorophyll with Apoproteins by Isolated Chloroplasts

The photosynthetic apparatus is reorganized during acclimation to various light environments. During adaptation of plants grown under a low-light to high-light environment, the light-harvesting chlorophyll a/b-protein complexes decompose concomitantly with an increase in the core complex of photosystem II. To study the mechanisms for reorganization of photosystems, the assembly of chlorophyll with apoproteins was investigated using isolated chloroplasts. When [14C]chlorophyllide b was incubated with chloroplasts in the presence of phytyl pyrophosphate, it was esterified and some of the [14C]chlorophyll b was converted to [14C]chlorophyll a via 7-hydroxymethyl chlorophyll. [14C]Chlorophyll a and b were incorporated into chlorophyll-protein complexes. Light-harvesting chlorophyll a/b-protein complexes of PSII had a lower [14C]chlorophyll a to [14C]chlorophyll b ratio than P700-chlorophyll a-protein complexes, indicating the specific binding of chlorophyll to apoproteins in our systems. 7-Hydroxymethyl chlorophyll, an intermediate molecule from chlorophyll b to chlorophyll a, did not become assembled with any apoproteins. These results indicate that chlorophyll b is released from light-harvesting chlorophyll a/b-protein complexes of photosystem II and converted to chlorophyll a via 7-hydroxymethyl chlorophyll in the lipid bilayer and is then used for the formation of core complexes of photosystems. These mechanisms provide the fast, fine regulation of the photosynthetic apparatus during construction of photosystems.     

Blue-Light-Regulated Expression of Genes for Two Early Steps of Chlorophyll Biosynthesis in Chlamydomonas reinhardtii

In light:dark-synchronized cultures of Chlamydomonas reinhardtii, the genes encoding the enzymes for two early steps of chlorophyll biosynthesis, glutamate-1-semialdehyde aminotransferase (gsa) and [delta]-aminolevulinic acid dehydratase (alad), are expressed at high levels early in the light phase, just prior to a rapid burst of chlorophyll synthesis. Induction of gsa mRNA in synchronized cells is totally dependent on light, whereas induction of alad mRNA occurs to approximately one-half the light-induced level even in cells kept in the dark during the light phase and appears to be dependent on the cell cycle or a circadian rhythm. gsa mRNA and alad mRNA accumulation is induced by light that was passed through blue (400-480 nm) or green (490-590 nm) filters but not by light that was passed through orange (>560 nm) or red (>610 nm) filters, indicating the participation of a blue-light photoreceptor system rather than a protochlorophyllide- or rhodopsin-based photoreceptor. Light induction of gsa mRNA accumulation is absent in a carotenoid-deficient mutant, which suggests that a carotenoid-containing blue-light photoreceptor is involved. In contrast, pretreatment of wild-type cells with either of two flavin antagonists, phenylacetic acid and KI, does not prevent the light induction. In the later part of the light phase, the gsa mRNA level decreases more rapidly than that of alad mRNA. Turnover studies indicate that the half-life of alad mRNA is twice that of gsa mRNA. This difference in mRNA stability partially accounts for the more rapid decline in gsa mRNA levels after the peak of light induction is reached. Thus, differential blue-light induction and stability of mRNAs regulates the expression of these two chlorophyll biosynthetic genes.     

Chlorophyll Content and the Pattern of Yellowing in Senescent Leaves

In senescent leaves of four species, leaf chlorophyll contentwas approximately linearly related to the estimated area ofyellow tissue, but the species differed in the amount of chlorophylllost before yellowing became apparent. Cotoneaster and Vicia leaves and Sinapis cotyledons, which produceddistinct yellowing patterns lost about 40 per cent, whereasSolanumleaves, which yellowed more uniformly lost over 60 percent of the chlorophyll before yellowing was visible. It issuggested that the leaf-cell population of Solanum may senescerather more synchronously than the other species, and that chlorophyllloss before yellowing may be a useful index of synchrony ofsenescence in leaves.     

衰老叶片和叶绿体中超氧阴离子和有机自由基浓度的变化

以电子自旋共振波谱技术,研究从水稻、玉米、花生和苋菜的成熟及衰老叶片分离的叶绿体中O_2~-的形成,以及干燥叶绿体和叶片粉末的有机自由基。经紫外光照射后老叶叶绿体的O_2~-浓度比成熟叶增大10～249％。冷冻干燥叶绿体及烘干叶片粉末在辐照前后皆有明显的单峰信号。辐照引发两种干样中形成更多的有机自由基。老叶制备的干样不论辐照或暗下都有较高水平的自由基。以上结果表明,叶片的衰老与自由基引起的损伤有关。     

Reduction of 2,6-dichlorophenol-indophenol (Hill Reaction) by Chloroplasts with Different Chlorophyll a/b Ratios Under monochromatic Light

Chloroplasts with different chlorophyll a/b ratios were isolated from 7 to 8 days old wheat seedlings and the activities of reduction of 2,6-dichlorophenol-indophenol (DPIP) by these chloroplasts as function of the chlorophyll a/b ratios were studied under mono-chromatic light (650 m μ, 670 mμ, 680 mμ, 707 mμ). It was found that the DPIP reducing activities by these chloroplasts varied with their chlorophyll a/b ratios, and these variations are affected by the wavelengths of the illuminating light. Under 650 mμ, at the a/b ratios of 2.2 to 2.82, the activities of DPIP reduction in- creased with the a/b ratios, but decreased when the a/b ratios were higher than 2.82. Under 670 mμ, the DPIP reducing activities also varied with the a/b ratios of isolated chloroplasts. However, the variation was more gradual and steadier. Under 680 mμ, the DPIP reducing activities increased with the a/b ratios over 3.0, but decreased rather suddenly at a/b ratio of 3.30. Essentially the same relation held for 707 mμ, but the Hill reaction activities ceased to decline farther when a/b ratio rose to 3.40 at 707 mμ. When the results were analyzed in terms of the “relative activities” of the chloroplasts of the above mentioned wave lengths, it was found that the values of the "relative activity" (a/a+b) declined steadily from 1.47–1.29 at an a/b ratio range of 2.05–3.40, while the values of the "relative activity" (b/a+b)increased steadily from 3.10--4.40 at the same range of a/b ratios. But it is to be noted that, under 650 mμ, the (a/a+b) was 1.36 at the a/b ratio range of 2.63–2.82. Interesting enough, the DPIP reducing activities were the highest of all with these a/b ratios. Activities of DPIP reduction by isolated chloroplasts kept at 0 ℃, 20 ℃, 30 ℃, and 45 ℃ diminished with time when illuminated at all wavelengths. However, for those kept at 45 ℃ their activities were lost after 20 minutes, except those illuminated with the wave length of 680 mμ which still maintained 30 % of the initial activity. When kept at the above mentioned temperatures, the chlorophyll a/b ratios of all batches of chloroplasts declined steadily with time. The above results are interpreted as being indicative of the possibility that the pigment systems for the partial reaction (Hill reaction) of the over-all photosynthesis process consisted mainly of Chlb650 and Chla670. And the correlation of temperature and abolition of activity reduction of DPIP of chloroplasts varied under monochromatic light of different wave lengths.     

Partial Purification and Characterization of Red Chlorophyll Catabolite Reductase,a Stroma Protein Involved in Chlorophyll Breakdown

Red chlorophyll (Chl) catabolite (RCC) reductase, which catalyzes the reaction of an intermediary Chl catabolite (RCC) in the two-step cleavage reaction of pheophorbide (Pheide) a into primary fluorescent catabolites (pFCCs) during Chl breakdown, was characterized and partially purified. RCC reductase activity was present at all stages of barley leaf development and even in roots. The highest specific activity was found in senescent leaves, which were used to purify RCC reductase 1000-fold. Among the remaining three proteins, RCC reductase activity was most likely associated with a 55-kD protein. RCC reductase exhibited saturation kinetics for RCC, with an apparent Michaelis constant of 0.6 mM. The reaction depended on reduced ferredoxin and was sensitive to oxygen. Assays of purified RCC reductase with chemically synthesized RCC as a substrate yielded three different FCCs, two of which could be identified as the stereoisomeric pFCCs from canola (Brassica napus) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. In the coupled reaction with Pheide a oxidase and RCC reductase, either pFCC-1 or pFCC-2 was produced, depending on the plant species employed as a source of RCC reductase. Data from 18 species suggest that the stereospecific action of RCC reductase is uniform within a plant family.     

Antimycin A Stimulation of Rate-limiting Steps of Photosynthesis in Isolated Spinach Chloroplasts

Changes in levels of metabolites in isolated spinach (Spinacia oleracea) chloroplasts seen upon addition of antimycin A suggest that the activities of enzymes mediating several regulated reactions are affected. Apparently, the presence of added antimycin A does not increase the level of CO₂ in the chloroplasts, nor does it stimulate CO₂ fixation by increasing the level of the carboxylation substrate, ribulose-1,5-diphosphate. Rather, it appears that antimycin A increases CO₂ fixation rate by indirectly stimulating the enzyme, ribulose-1,5-diphosphate carboxylase (E.C. 4.1.1.39), which mediates the carboxylation of ribulose-1,5-diphosphate to give 3-phosphoglycerate. Another rate-limiting enzyme of the reductive pentose phosphate cycle, hexose diphosphatase (E.C. 3.1.3.11), seems also to be stimulated. The synthesis of polysaccharides (mostly starch) seems also to be stimulated. These results are interpreted as indicating that antimycin A addition enhances the general activation of those enzymes which already are activated during photosynthesis but are inactive in the dark. The ratio of adenosine triphosphate-adenosine diphosphate under conditions of photosynthesis was only moderately decreased in the presence of antimycin A, perhaps accounting in part for an observed increase in accumulation of 3-phosphoglycerate as compared with dihydroxyacetone phosphate. No significant effect on movement of metabolites from the chloroplast to the medium was seen.     

Synthesis and Breakdown of the Apoproteins of Light-Harvesting Chlorophyll a/b Proteins in Chlorophyll b-deficient Mutants of Rice

Turnover, in the light, of apoproteins of light-harvesting chlorophylla/6-proteins for Photo-system I and II (LHC-I and LHC-II, respectively)was studied with the wild-type and three chlorophyll 6-deficientmutants of rice. (1) Synthesis of the 24 and 25 kDa apoproteinsof LHC-II and the 20 and 21 kDa apoproteins of LHC-I was examinedby incubating leaf segments with [³⁵S]-methionine. The threerice mutants, chlorina 2, which totally lacks chlorophyll b,and chlorina 11 and 14, which are partially deficient in chlorophyllb, synthesized the apoproteins as rapidly as did the wild typerice. (2) Pulse-chase experiments showed that breakdown of theapoproteins proceeded slowly, such that only a small proportionof the newly synthesized apoproteins was lost during the 48h of the chase in normal rice leaves. By contrast, large fractionsof the labelled apoproteins were rapidly degraded within thefirst several hours of the chase period in the chlorina mutants.The greater the deficiency in chlorophyll b of the mutant, thelarger were the rate and extent of the protein breakdown. Thisresult indicates that chlorophyll b is needed to stabilize theapoproteins of LHC-II and LHC-I. (3) However, even in chlorina2, there were small fractions of the apoproteins with lifetimesas long as those of apoproteins in the wild-type rice, suggestingthat the newly synthesized apoproteins are partially protectedby a factor(s) other than chlorophyll b. (4) The rate of turnoverof the apoproteins was significantly reduced in the dark andstrongly inhibited by prior treatment of leaf segments withchloramphenicol. (Received November 24, 1988; Accepted March 17, 1989)     

Rate-Limiting Steps of Electron Transport in Chloroplasts during Ontogeny and Senescence of Barley

Partial photochemical activities and concentrations of electron carriers were measured relative to chlorophyll in barley (Hordeum vulgare L.) thylakoids, isolated from primary leaves during ontogeny and senescence. Thylakoids from mature leaves generated somewhat higher quantum efficiencies than thylakoids from premature or senescing leaves; this phenomenon did not appear to be caused by any deficiency of water-splitting enzyme. Under conditions of saturating light, the noncyclic electron flux from water to the reducing side of photosystem I increased during leaf ontogeny, peaked at maturity, and declined during senescence. However, electron fluxes appeared to be limited at different steps before and after leaf maturity. Before leaf maturity, the rate-limiting step was located prior to the reoxidation of plastohydroquinone. After leaf maturity, the decline in noncyclic electron flux correlated with a decrease in the concentration of cytochromes f and b₆. This correlation, together with a consideration of mechanisms of entry and exit of electrons in 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated thylakoids, suggests that the cytochrome f/b₆-containing complex, and not plastocyanin or P700, is the site of entry of electrons from the reduced forms of 2,6-dichlorophenolindophenol and diaminodurene. It is therefore proposed that in senescing leaves the cytochrome f/b₆-containing complex limited electron transport by constraining the rate of reduction of cytochrome f by plastohydroquinone.     

Supramolecular Assembly and Function of Subunits Associated with the Chlorophyll a-b Light-Harvesting Complex II (LHC II) in Soybean Chloroplasts

The chlorophyll (Chl) a-b light harvesting complex II (LHC II)contains more than 80% of the light-harvesting pigments of photosystemII (PS II) in chloroplasts. The supramolecular assembly andfunction of this auxiliary antenna system was investigated inChi b-deficient and Chi b-less mutant chloroplasts from soybeanand barley plants, and in their wild-type counterparts. Fourdistinct LHC II polypeptides were resolved by SDS-PAGE (subunitsa, b, c and d), having apparent molecular masses of 29, 28,27.2 and 26.8 kDa, respectively. The analysis of LHC II subunitcomposition in different developmental stages of the PS II unitin soybean (3>Chla/Chlbb>6), indicated the associationof specific subunits with the LHC H-inner and LHC II-peripheralin the chloroplast. The amount of subunit a in PS II was constantover a broad range of Chl a/Chl b ratios, suggesting that thissubunit is closely associated with the PS II-core complex. Subunitd also appeared to be constant over a wide range of Chl a/Chlb ratios, suggesting close association with the LHC II-inner.The PS II content in subunits b and c increased with the PSII antenna development in soybean but the ratio of b/c remainedconstant in all developmental stages and equal to 2 :1. Subunita was present in the Chl b-less chlorina f2 mutant of barleygrown under continuous illumination but was absent under intermittentillumination. The results suggest that each subunit binds 13-15Chl molecules. A working hypothesis is presented on the PS IIantenna development and LHC II subunit composition in soybeanchloroplasts. (Received October 11, 1988; Accepted January 19, 1989)     

Substitution of Cytokinins and Chlorophyll Breakdown in Detached Wheat Leaves

Four derivatives of benzyladenine with alkyl-substitution atthe 9 position, (9-methyl, 9-methoxymethyl, 9-tetrahydro-2-pyranyland 9-cyclopentyl-N⁶ benzyladenine), can slow, depending uponthe substituent and concentration, the breakdown of chlorophyllin detached wheat leaves. (Received June 9, 1984; Accepted September 25, 1984)