Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat

Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.     

Influence of photodynamic processes induced by 2,2′-dipyridyl on the enzymatic system of chlorophyll biosynthesis

The influence of 2,2′-dipyridyl (2,2′-DP) on the activity of one of the enzymes at the initial stages of chlorophyll (Chl) biosynthesis, δ-aminolevulinic acid dehydratase (ALAD; δ-aminolevulinate hydro-lyase, EC 4.2.1.24), as well as on δ-aminolevulinic acid (ALA) accumulation was investigated in green barley (Hordeum vulgare L.) leaves. In seven-day-old green leaves treated with 3 mM 2,2′-DP for 17 h in darkness and subsequently irradiated with "white light" (15 W m-2) for 4, 8, and 24 h the ALAD activity was 51 % as compared to that in untreated leaves. At the same time, the ALA forming system was most sensitive to the photodynamic processes caused by 2,2′-DP. After 8 h of irradiation, ALA synthesis was entirely inhibited. After the treatment the leaves accumulated exceptionally high amounts of Chl precursors such as protoporphyrin IX (Proto), Mg-protoporphyrin IX (Mg-Proto), its monomethyl ester, and protochlorophyllide (Pchlide) that are photosensitizers of photodynamic processes in plants. A comparatively low Chl and carotenoid (Car) destruction was registered during the subsequent 4 and 8 h of irradiation. At the same time, the content of Chl precursors was negligible. The low photodestruction of Chl and Car included in pigment-protein complexes, against the background of fast porphyrin disappearance, and fast decrease of enzymatic activities at the initial stages of Chl production could mean that the photodynamic effect induced by porphyrins accumulated in the presence of 2,2′-DP affected first the Chl enzymatic system and did not change the pool of already synthesized photosynthetic pigments.     

Salt‐stress induced modulation of chlorophyll biosynthesis during de‐etiolation of rice seedlings

Chlorophyll biosynthesis in plants is subjected to modulation by various environmental factors. To understand the modulation of the chlorophyll (Chl) biosynthesis during greening process by salt, 100–200 mM NaCl was applied to the roots of etiolated rice seedlings 12 h prior to the transfer to light. Application of 200 mM NaCl to rice seedlings that were grown in light for further 72 h resulted in reduced dry matter production (–58%) and Chl accumulation (–66%). Ionic imbalance due to salinity stress resulted in additional downregulation (41–45%) of seedling dry weight, Chl and carotenoid contents over and above that of similar osmotic stress induced by polyethylene glycol. Downregulation of Chl biosynthesis may be attributed to decreased activities of Chl biosynthetic pathway enzymes, i.e. 5‐aminolevulinic acid (ALA) dehydratase (EC‐2.4.1.24), porphobilinogen deaminase (EC‐4.3.1.8), coproporphyrinogen III oxidase (EC‐1.3.3.3), protoporphyrinogen IX oxidase (EC‐1.3.3.4), Mg‐protoporphyrin IX chelatase (EC‐6.6.1.1) and protochlorophyllide oxidoreductase (EC‐1.3.33.1). Reduced enzymatic activities were due to downregulation of their protein abundance and/or gene expression in salt‐stressed seedlings. The extent of downregulation of ALA biosynthesis nearly matched with that of protochlorophyllide and Chl to prevent the accumulation of highly photosensitive photodynamic tetrapyrroles that generates singlet oxygen under stress conditions. Although, ALA synthesis decreased, the gene/protein expression of glutamyl‐tRNA reductase (EC‐1.2.1.70) increased suggesting it may play a role in acclimation to salt stress. The similar downregulation of both early and late Chl biosynthesis intermediates in salt‐stressed seedlings suggests a regulatory network of genes involved in tetrapyrrole biosynthesis.     

A short overview of chlorophyll biosynthesis in algae

Chlorophylls are the most abundant classes of natural pigments and their biosynthesis is therefore a major metabolic activity in the ecosphere. Two pathways exist for chlorophyll biosynthesis, one taking place in darkness and the other requiring continuous light as a precondition. The key process for Chl synthesis is the reduction of protochlorophyllide (Pchlide). This enzymatic reaction is catalysed by two different enzymes — DPOR (dark-operative Pchlide oxidoreductase) or the structurally distinct LPOR (light-dependent Pchlide oxidoreductase). DPOR which consists of three subunits encoded by three plastid genes in eukaryotes was subject of our study. A short overview of our present knowledge of chlorophyll biosynthesis in Chlamydomonas reinhardtii in comparison with other plants is presented. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Biosynthesis of delta-aminolevulinic acid and the regulation of heme formation by immature erythroid cells in man

Heme formation in the erythron is subject to end product regulation by negative feedback, but the exact point of metabolic control in human erythroid cells is unknown. To investigate the mode of action of heme on its own formation, the effects of micromolar concentrations of hemin on de novo synthesis of protoporphyrin IX and delta-aminolevulinate (delta-ALA) by intact human reticulocytes were examined in the presence of 1 mM alpha,alpha'-bipyridyl and 200 microM 4,6-dioxoheptanoate to block their further conversion by ferrochelatase or delta-ALA dehydrase, respectively. At final concentrations (25-40 microM), hemin, which is known to reduce incorporation of [2-14C]glycine into cellular heme, significantly inhibited formation of protoporphyrin IX and total delta-aminolevulinate in situ by these cells. Since synthesis of the first committed precursor, delta-aminolevulinate, as well as protoporphyrin (which is derived from it) were diminished, the effects of hemin on delta-aminolevulinate synthase (EC 2.3.1.37) were studied. Hemin, at concentrations up to 40 microM, had no direct effect on enzymatic activity, as measured with [5-14C] alpha-ketoglutarate (in hypotonically lysed cells) or [1,4-14C]succinyl coenzyme A (in deoxycholate lysates), even after preincubation. However, when intact human reticulocytes were incubated with hemin before assay for delta-ALA synthase, there was a rapid, concentration-dependent reduction in enzymatic activity (mean 42 and 23% inhibition after 60 min for these two substrates, respectively). Hemin had no effect on steady-state levels of delta-ALA synthase mRNA, as determined by Northern blot hybridization using an erythroid-specific human cDNA probe. Thus, a mechanism for inducing feedback inhibition of the tetrapyrrole pathway exists in human erythroid cells. It controls formation of the first committed precursor of protoporphyrin IX, delta-aminolevulinate, and hence regulates heme biosynthesis by limiting the availability of the porphyrin, rather than the metal substrate for the ferrochelatase reaction. Hemin interacts with constituents of the intact reticulocyte significantly to reduce delta-aminolevulinic acid synthase activity by an indirect cellular process that does not influence the abundance of erythroid-specific synthase mRNA but may either inhibit its ribosomal translation in an unknown manner or promote degradation of the enzyme itself by specific proteolysis.     

Herbivorous insects alter the chlorophyll metabolism of galls on host plants

Five types of insect-induced galls derived from three host plant leaves were analyzed for their carotenoid (Car), chlorophyll (Chl), and Chl biosynthesis porphyrins such as protoporphyrinogen IX (PPIX), magnesium protoporphyrin (MGPP) and protochlorophyllide (Pchlide), and Chl degradation intermediates including chlorophyllide (Chlide), pheophytin (Phe), pheophorbide (Pho), and phytylated and dephytylated pigments, and compared to ungalled portions of the same leaf. Galls contain significantly lower levels of Chl-related compounds (CRCs) than ungalled portions of host leaves. The mole percent of porphyrin and the ratios of Chlide/Phe and phytylated/dephytylated pigments are both very different between galls and host leaves. We, therefore, conclude that leaf-derived gall is a kind of non-leaf green tissue, that herbivorous insects alter gall Chl biosynthesis and degradation pathways, that Mg-chelatase, Mg-dechelatase, and chlorophyllase may be the major non-lethal enzymes in galls, and that while ungalled host leaves take Chl → Phe → Pho and Chl → Chlide → Pho as the major and minor degradation routes, respectively, all galls are in contrast with the host leaves.     

Effects of iron deficiency on heme biosynthesis in Rhizobium japonicum.

The effects of iron deficiency on heme biosynthesis in Rhizobium japonicum were examined. Iron-deficient cells had a decreased maximum cell yield and a decreased cytochrome content and excreted protoporphyrin into the growth medium. The activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (EC 2.3.1.37) and delta-aminolevulinic acid dehydrase (EC 4.2.1.24), were diminished in iron-deficient cells, but were returned to normal levels upon addition of iron to the cultures. The addition of iron salts, iron chelators, hemin, or protoporphyrin to cell-free extracts did not affect the activity of these enzymes. The addition of levulinic acid to iron-deficient cultures blocked protoporphyrin excretion and also resulted in high delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase activities. These results suggest the possibility that rhizobial heme biosynthesis in the legume root nodule may be affected by the release of iron from the host plant to the bacteroids.     

Light-independent accumulation of essential chlorophyll biosynthesis- and photosynthesis-related proteins in <Emphasis Type="Italic">Pinus mugo</Emphasis> and <Emphasis Type="Italic">Pinus sylvestris</Emphasis> seedlings

Dark-grown seedlings of Pinus mugo Turra and Pinus sylvestris L. accumulate chlorophyll (Chl) and its precursor protochlorophyllide (Pchlide). Pchlide reduction is a key regulatory step in Chl biosynthesis. In the dark, Pchlide is reduced by light-independent Pchlide oxidoreductase (DPOR) encoded by three plastid genes chlL, chlN, and chlB (chlLNB). To investigate the differences in chlLNB gene expressions, we compared the dark-grown and 24-h illuminated seedlings of P. mugo and P. sylvestris. Expression of these genes was found constitutive in all analyzed samples. We report light-independent accumulation of important proteins involved in Chl biosynthesis (glutamyl-tRNA reductase) and photosystem formation (D1 and LHCI). Chl and Pchlide content and plastid ultrastructure studies were also performed.     

宽叶吊兰叶绿素生物合成的昼夜节律变化

在被子植物中,从谷氨酰-tRNA到叶绿素的生物合成是由许多酶催化的级联反应,其中间代谢产物具有较强的光反应活性和细胞毒性,因此这一过程在细胞内受到严格的调控。本研究通过检测宽叶吊兰叶片叶绿素生物合成途径的14种中间产物含量随昼夜节律的变化,探讨昼夜节律对宽叶吊兰叶绿素生物合成的影响。结果表明,中间产物ALA(δ-氨基乙酰丙酸)、PBG(胆色素原)、ProtoⅨ(原卟啉Ⅸ)、Heme(血红素)、Mg-ProtoⅨ(镁原卟啉Ⅸ)、Chlide a(叶绿素酸酯a)、Chlide b(叶绿素酸酯b)、Chl a(叶绿素a)、Chl b(叶绿素b)受光诱导,而UrogenⅢ(尿卟啉Ⅲ)、CoprogenⅢ(粪卟啉Ⅲ)和Pchlide(原叶绿素酸脂)受黑暗诱导,尤其是Pchlide在黑暗中的积累量显著增加;Mpe(镁原卟啉Ⅸ单甲酯)和Mpde(镁原卟啉Ⅸ二酯)具有2个积累峰值,分别出现在中午12∶00和夜间24∶00。说明叶绿素生物合成受昼夜节律的调控,但其中间代谢产物含量的变化规律与昼夜节律并不完全一致。     

Cytokinin effects on tetrapyrrole biosynthesis and photosynthetic activity in barley seedlings

Cytokinin promotes morphological and physiological processes including the tetrapyrrole biosynthetic pathway during plant development. Only a few steps of chlorophyll (Chl) biosynthesis, exerting the phytohormonal influence, have been individually examined. We performed a comprehensive survey of cytokinin action on the regulation of tetrapyrrole biosynthesis with etiolated and greening barley seedlings. Protein contents, enzyme activities and tetrapyrrole metabolites were analyzed for highly regulated metabolic steps including those of 5-aminolevulinic acid (ALA) biosynthesis and enzymes at the branch point for protoporphyrin IX distribution to Chl and heme. Although levels of the two enzymes of ALA synthesis, glutamyl-tRNA reductase and glutamate 1-semialdehyde aminotransferase, were elevated in dark grown kinetin-treated barley seedlings, the ALA synthesis rate was only significantly enhanced when plant were exposed to light. While cytokinin do not stimulatorily affect Fe-chelatase activity and heme content, it promotes activities of the first enzymes in the Mg branch, Mg protoporphyrin IX chelatase and Mg protoporphyrin IX methyltransferase, in etiolated seedlings up to the first 5 h of light exposure in comparison to control. This elevated activities result in stimulated Chl biosynthesis, which again parallels with enhanced photosynthetic activities indicated by the photosynthetic parameters F _V/F _M, J _CO2max and J _CO2 in the kinetin-treated greening seedlings during the first hours of illumination. Thus, cytokinin-driven acceleration of the tetrapyrrole metabolism supports functioning and assembly of the photosynthetic complexes in developing chloroplasts.     

Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana.

Illumination releases the arrest in chlorophyll (Chl) biosynthesis in etiolated angiosperm seedlings through the enzymatic photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the first light-dependent step in chloroplast biogenesis. NADPH: Pchlide oxidoreductase (POR, EC 1.3.1.33), a nuclear-encoded plastid-localized enzyme, mediates this unique photoreduction. Paradoxically, light also triggers a drastic decrease in the amounts of POR activity and protein before the Chl accumulation rate reaches its maximum during greening. While investigating this seeming contradiction, we identified two distinct Arabidopsis thaliana genes encoding POR, in contrast to previous reports of only one gene in angiosperms. The genes, designated PorA and PorB, by analogy to the principal members of the phytochrome photoreceptor gene family, display dramatically different patterns of light and developmental regulation. PorA mRNA disappears within the first 4 h of greening, whereas PorB mRNA persists even after 16 h of illumination, mirroring the behavior of two distinct POR protein species. Experiments designed to help define the functions of POR A and POR B demonstrate exclusive expression of PorA in young seedlings and of PorB both in seedlings and in adult plants. Accordingly, we propose the existence of a branched light-dependent Chl biosynthesis pathway in which POR A performs a specialized function restricted to the initial stages of greening and POR B maintains Chl levels throughout angiosperm development.     

Plastid development in germinating wheat (Triticum aestivum) is enhanced by gibberellic acid and delayed by gabaculine

Etioplast development and protochlorophyllide (Pchlide) accumulation was studied in wheat seedlings ( Triticum aestivum L. cv. Walde, Weibull) grown in darkness on gibberellic acid (GA₃), gabaculine (3-amino-2,3-dihydrobenzoic acid), or on a combination of the two. The results were compared with the features of seedlings grown on water only. GA₃ enhanced shoot growth and promoted etioplast development. A correlation was observed between the appearance of prolamellar bodies (PLBs) and of phototransformable Pchlide. Gabaculine, a known tetrapyrrole biosynthesis inhibitor, delayed growth, slowed down the rate of PLB formation and caused structural alterations of the etioplasts up to 48 h of germination. Gabaculine also delayed the formation of phototransformable Pchlide as well as overall Pchlide biosynthesis, as determined by low-temperature fluorescence emission in vivo. The spectral blue-shift of newly formed chlorophyllide (Chlide) was delayed in irradiated dark-grown gabaculine-grown seedlings, indicating an inhibited dissociation of Chlide and NADPH-Pchlide oxidoreductase (Pchlide reductase: EC 1.3.1.33). Thus there is a close correlation between accumulation of Pchlide and etioplast development, also under conditions when development is enhanced or delayed.     

Evidence of chlorophyll synthesis pathway alteration in desiccated barley leaves

In etiolated leaves, saturating flash of 200 ms induces phototransformation of protochlorophyllide (Pchlide) F655 into chlorophyllide (Chlide), then into Chl through reactions which do not need light sensibilisation. The synthesis of Chl is known to be slowed down in etiolated leaves exposed to desiccation stress. In order to analyse the intensity and time-course of Chlide transformation into Chl, we used the fluorescence emission of etiolated leaves previously exposed to a 200 ms saturating flash. We used low-temperature fluorescence spectroscopy to reveal the inhibition site of Chl synthesis in etiolated barley leaves exposed to water stress. Shibata shift appears as the main target point of the water deficit. It was found that water deficit inhibits partially active Pchlide F655 regeneration. Also, esterification of Chlide into Chl is impaired. It appears that these inhibitory effects alter the appearance of PSII active reaction centres.     

Enzymatic Activities for the Synthesis of Chlorophyll in Pigment-Deficient Variegated Leaves of Euonymus japonicus

The enzymes involved in the biosynthesis of chlorophyll (Chl)in pigment-deficient variegated leaves of Euonymus japonicuswere investigated. Each variegated leaf was composed of clearlydelineated green and white sectors. The white sectors containedalmost no Chls. The rate of synthesis of 5-aminolevulinic acid(ALA) in the white sectors in vivo was twice that in the greensectors. The level of glutamate 1-semialdehyde aminotransferasein the white sectors was much higher than that in the greensectors. Plastidic tRNA^Glu was also present at substantial levelsin the white sectors, indicating that the system for synthesisof ALA was very active in the white sectors. The activity of porphobilinogen (PBG) synthase in the whitesectors in vitro was twice that in the green sectors. In thewhite sectors the rate of porphyrin synthesis from PBG was 4-to 6-fold higher than in the green sectors. We measured Mg-chelataseactivity indirectly in both sectors by monitoring the accumulationof Mg-protoporphyrin IX in the presence of 2,2'-dipyridyl, whichinhibits isocyclic ring formation with the resultant accumulationof Mg-protoporphyrin IX. When sectors were incubated in darknesswith 2,2'-dipyridyl, large amounts of protoporphyrin IX accumulatedin the white sectors, whereas Mg-protoporphyrin IX mainly accumulatedin the green sectors. These results suggest that the enzymesfor the synthesis of porphyrin that catalyze conversion of ALAto protoporphyrin IX were very active and that the Mg-insertionstep might be blocked in the white sectors, with the resultantfailure to synthesize Chl. The deficiency is discussed in acomparison with that in other Chl-deficient plants. (Received November 15, 1995; Accepted March 21, 1996)     

Cerium Relieves the Inhibition of Chlorophyll Biosynthesis of Maize Caused by Magnesium Deficiency

Lanthanoids (Ln) were demonstrated to improve chlorophyll formation and the growth of plants. But the mechanism of the fact that Ln promotes chlorophyll biosynthesis of plants is poorly understood. The main aim of the study was to determine Ln effects in chlorophyll formation of maize under magnesium (Mg) deficiency. Maize plants were cultivated in Hoagland’s solution. They were subjected to Mg deficiency and to cerium administered in Mg-deficient Hoagland’s media, and then the contents of various chlorophyll precursors and gen expressions of the key enzymes of chlorophyll biosynthesis were examined. The decrease of chlorophyll contents in maize leaves caused by Mg deficiency suggested an inhibition of chlorophyll synthesis that was inhibited by a reduction of the precursors as measured by analyzing the contents of δ-aminolevulinic acid, porphobilinogen, uroporphyrinogen III, Mg–protoporphyrin IX, and protochlorophyll, as well as the expression levels of magnesium chelatase, magnesium-protoporphyrin IX methyltransferase, and chlorophyll synthase; Mg deficiency significantly inhibited the transformation from coproporphyrinogen III or protoporphyrin IX to chlorophyll. However, cerium addition significantly relieved the inhibition of chlorophyll biosynthesis in maize caused by Mg deficiency and increased chlorophyll content and promoted a series of transformations from δ-aminolevulinic acid to chlorophyll and maize growth under Mg deficiency. It implied that cerium might partly substitute for the role of Mg.     

Regeneration of protochlorophyllide in green and greening leaves of plants with varying proportions of protochlorophyllide forms in darkness

During illumination of dark-grown plants protochlorophyllide (Pchlide) is continuously transformed to chlorophyllide (Chlide). Different dark-grown plants, maize ( Zea mays cv. Sundance), wheat ( Triticum aestivum cv. Kosack), pea ( Pisum sativum cv. Kelwedon wonder), the lip1 mutant of pea, and the aurea mutant of tomato ( Solanum lycopersicum ), have various ratios of spectral Pchlide forms in darkness. When the plants were illuminated and then returned to darkness Pchlide re-accumulated. The proportions of different Pchlide forms within the pool of re-accumulated Pchlide were followed by low temperature fluorescence emission and excitation spectra in green and greening leaves. After 1 h of illumination the spectral characteristics of regenerated Pchlide forms mirrored those of Pchlide in dark-grown plants and were thus species dependent. After a prolonged illumination period (24 h) as well as in fully green leaves energy transfer to chlorophyll (Chl) masked the presence of long-wavelength Pchlide in the fluorescence emission spectra. However, excitation spectra showed Pchlide absorption around 650 nm and its flash-induced disappearance confirmed its nature of phototransformable Pchlide. In fact the excitation spectra showed that the proportions of different Pchlide forms in green leaves highly resembled the proportions of Pchlide forms in dark-grown leaves and were specific for the plant variety. Thus Chl formation in both dark-grown and light-grown leaves can occur in a similar way through the main photoactive long-wavelength form of Pchlide.     

The C-terminal extension of ferrochelatase is critical for enzyme activity and for functioning of the tetrapyrrole pathway in Synechocystis strain PCC 6803

Heme and chlorophyll (Chl) share a common biosynthetic pathway up to the branch point where magnesium chelatase and ferrochelatase (FeCH) insert either magnesium for Chl biosynthesis or ferrous iron for heme biosynthesis. A distinctive feature of FeCHs in cyanobacteria is their C-terminal extension, which forms a putative transmembrane segment containing a Chl-binding motif. We analyzed the ΔH324 strain of Synechocystis sp. strain PCC 6803, which contains a truncated FeCH enzyme lacking this C-terminal domain. Truncated FeCH was localized to the membrane fraction, suggesting that the C-terminal domain is not necessary for membrane association of the enzyme. Measurements of enzyme activity and complementation experiments revealed that the ΔH324 mutation dramatically reduced activity of the FeCH, which resulted in highly upregulated 5-aminolevulinic acid synthesis in the ΔH324 mutant, implying a direct role for heme in the regulation of flux through the pathway. Moreover, the ΔH324 mutant accumulated a large amount of protoporphyrin IX, and levels of Chl precursors were also significantly increased, suggesting that some, but not all, of the “extra” flux can be diverted down the Chl branch. Analysis of the recombinant full-length and truncated FeCHs demonstrated that the C-terminal extension is critical for activity of the FeCH and that it is strictly required for oligomerization of this enzyme. The observed changes in tetrapyrrole trafficking and the role of the C terminus in the functioning of FeCH are discussed.     

Characterization of a family of chlorophyll-deficient wheat (Triticum) and barley (Hordeum vulgare) mutants with defects in the magnesium-insertion step of chlorophyll biosynthesis.

During thylakoid membrane biogenesis, chlorophyll (Chl) biosynthesis and the accumulation of Chl-binding proteins are tightly linked, light-regulated processes. We have investigated the consequences faced by mutant plants with defects in Chl biosynthesis by studying a series of five homeologous allelic chlorina mutants in wheat (Triticum) and one phenotypically related barley (Hordeum vulgare) mutant that express the same pleiotropic mutant phenotype but to different extents. These mutants accumulate Chl at different rates, with the most severely affected plants having the slowest rate of Chl accumulation. Analysis of precursor pools in the Chl synthesis pathway indicates they have a partial block in Chl synthesis and accumulate protoporphyrin IX (Proto), the last porphyrin compound common to both heme and Chl synthesis. The affected plants with the most severe phenotypes accumulate the most Proto. Chloroplasts isolated from these mutants exhibit a lower activity of the enzyme Mg-chelatase, which catalyzes the first committed step in Chl synthesis. The most severely affected plants exhibit the greatest reduction in Mg-chelatase activity. Heme levels and protoporphyrinogen oxidase activity were the same for mutant and wild-type plants. We suggest that a block in Mg-chelatase activity in these mutants could account for the other traits of their pleiotropic phenotype previously described in the literature.