Arabidopsis light-dependent protochlorophyllide oxidoreductase A (PORA) is essential for normal plant growth and development

During skotomorphogenesis in angiosperms, NADPH:protochlorophyllide oxidoreductase (POR) forms an aggregate of photolabile NADPH-POR-protochlorophyllide (Pchlide) ternary complexes localized to the prolamellar bodies within etioplasts. During photomorphogenesis, POR catalyzes the light-dependent reduction of Pchlide a to chlorophyllide (Chlide) a, which is subsequently converted to chlorophyll (Chl). In Arabidopsis there are three structurally related POR genes, denoted PORA, PORB and PORC. The PORA and PORB proteins accumulate during skotomorphogenesis. During illumination, PORA is only transiently expressed, whereas PORB and PORC persist and are responsible for bulk Chl synthesis throughout plant development. Here we have tested whether PORA is important for skotomorphogenesis by assisting in etioplast development, and normal photomorphogenic development. Using reverse genetic approaches, we have identified the porA-1 null mutant, which contains an insertion of the maize Dissociation transposable element in the PORA gene. Additionally, we have characterized PORA RNAi lines. The porA-1 and PORA RNAi lines display severe photoautotrophic growth defects, which can be partially rescued on sucrose-supplemented growth media. Elimination of PORA during skotomorphogenesis results in reductions in the volume and frequency of prolamellar bodies, and in photoactive Pchlide conversion. The porA-1 mutant characterization thus establishes a quantitative requirement for PORA in etioplast development by demonstrating significant membrane ultrastructural and biochemical defects, in addition to suggesting PORA-specific functions in photomorphogenesis and plant development.     

Arabidopsis protochlorophyllide oxidoreductase A (PORA) restores bulk chlorophyll synthesis and normal development to a porB porC double mutant

In angiosperms the strictly light-dependent reduction of protochlorophyllide to chlorophyllide is catalyzed by NADPH:protochlorophyllide oxidoreductase (POR). The Arabidopsis thaliana genome encodes three structurally related but differentially regulated POR genes, PORA, PORB and PORC. PORA is expressed primarily early in development—during etiolation, germination and greening. In contrast, PORB and PORC are not only expressed during seedling development but also throughout the later life of the plant, during which they are responsible for bulk chlorophyll synthesis. The Arabidopsis porB-1 porC-1 mutant displays a severe xantha (highly chlorophyll-deficient) phenotype characterized by smaller prolamellar bodies in etioplasts and decreased thylakoid stacking in chloroplasts. Here we have demonstrated the ability of an ectopic PORA overexpression construct to restore prolamellar body formation in the porB-1 porC-1 double mutant background. In response to illumination, light-dependent chlorophyll production, thylakoid stacking and photomorphogenesis are also restored in PORA-overexpressing porB-1 porC-1 seedlings and adult plants. An Arabidopsis porB-1 porC-1 double mutant can therefore be functionally rescued by the addition of ectopically expressed PORA, which suffices in the absence of either PORB or PORC to direct bulk chlorophyll synthesis and normal plant development.     

Substrate-dependent and organ-specific chloroplast protein import in planta

The NADPH-dependent protochlorophyllide (Pchlide) oxidoreductase (POR) is unique because it is a photoenzyme that requires light for its catalytic activity and uses Pchlide itself as a photoreceptor. In Arabidopsis, there are three structurally related PORs, denoted PORA, PORB, and PORC. The import of one of them, PORA, into plastids of cotyledons is substrate dependent. This substrate dependence is demonstrated in intact seedlings of wild-type Arabidopsis and two mutants, xantha2, which is devoid of Pchlide, and flu, which upon redarkening rapidly accumulates Pchlide. In true leaves, PORA uptake does not require the presence of Pchlide. The organ specificity of the substrate-dependent import of PORA reveals a means of controlling plastid protein translocation that is closely associated with a key step in plant development, the light-dependent transformation of cotyledons from a storage organ to a photosynthetically active leaf.     

Overexpression of light-dependent PORA or PORB in plants depleted of endogenous POR by far-red light enhances seedling survival in white light and protects against photooxidative damage

The structurally related light-dependent protochlorophyllide (Pchlide) oxidoreductases PORA and PORB mediate the only light-requiring step in chlorophyll (Chl) biosynthesis in higher plants. Correlative evidence suggests that some in vivo functions of PORA and PORB may be unique, including a postulated photoprotective role for PORA. For example, wild-type Arabidopsis thaliana seedlings grown in non-photooxidative far-red light (cFR) resemble those grown in white light (WL), but they are yellow and do not green normally thereafter in WL. This defect is accompanied by the absence of detectable PORA and reduced levels of PORB expression. Here, direct evidence is provided that the presence of POR, either as PORA or PORB, can confer photoprotection in plants. In contrast to the wild-type, the plastids of transgenic PORA- or PORB-overexpressing Arabidopsis seedlings grown in cFR possess extensive prolamellar bodies. Upon a subsequent shift to WL, POR-overexpressing seedlings develop thylakoid membranes, accumulate large amounts of Chl and are viable at fluence rates lethal to the wild-type. Intriguingly, the plastid membrane architectures of greening transgenic seedlings seem to depend on whether PORA or PORB has been overproduced. POR-overexpressing seedlings shifted from cFR to WL of fluence rates from 20 to 500 μE m^–2 sec^–1 accumulate substantially higher amounts of Chl than does the wild-type. Furthermore, the WL fluence rate that permits maximal Chl accumulation increases from 8 μE m^–2 sec^–1 in the wild-type to 125 μE m^–2 sec^–1 in transgenic seedlings. POR overexpression during growth in cFR also correlates with a fourfold decrease in the steady-state content of Pchlide, a potentially lethal photosensitizer.     

Photoactive Protochlorophyllide-Enzyme Complexes Reconstituted with PORA,PORB and PORC Proteins of A. thaliana: Fluorescence and Catalytic Properties

Photoactive Pchlide-POR-NADPH complexes were reconstituted using protochlorophyllide (Pchlide) and recombinant light-dependent protochlorophyllide oxidoreductase (POR) proteins, His₆-PORA, His₆-PORB and His₆-PORC, from Arabidopsis thaliana. We did not observe any differences in the kinetics of the protochlorophyllide photoreduction at room temperature among the PORA, PORB and PORC proteins. In contrast, the PORC protein showed lower yield of Chlide formation than PORA and PORB when preincubated in the dark for 30 min and then illuminated for a short time. The most significant observation was that reconstituted Pchlide-POR-NADPH complexes showed fluorescence maxima at 77 K similar to those observed for highly aggregated Pchlide-POR-NADPH complexes in prolamellar bodies (PLBs) in vivo. Homology models of PORA, PORB and PORC of Arabidopsis thaliana were developed to compare predicted structures of POR isoforms. There were only slight structural differences, mainly in the organisation of helices and loops, but not in the shape of whole molecules. This is the first comparative analysis of all POR isoforms functioning at different stages of A. thaliana development.     

Etioplast differentiation in arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant.

The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide-F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide-F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide-F655 to nonphotoactive Pchl[ide]-F632, and the amount of Pchlide-F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of etioplasts containing an extensive PLB and large amounts of photoactive Pchlide-F655.     

Functional analysis of isoforms of NADPH: protochlorophyllide oxidoreductase (POR), PORB and PORC, in Arabidopsis thaliana

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide. To elucidate the physiological function of three differentially regulated POR isoforms (PORA, PORB and PORC) in Arabidopsis thaliana, we isolated T-DNA tagged null mutants of porB and porC. The mature seedlings of the mutants had normal photosynthetic competencies, showing that PORB and PORC are interchangeable and functionally redundant in developed plants. In etiolated seedlings, only porB showed a reduction in the photoactive protochlorophyllide and the size of prolamellar bodies (PLBs), indicating that PORB, as well as PORA, functioned in PLB assembly and photoactive protochlorophyllide formation in etiolated seedlings. When illuminated, the etiolated porB seedling was able to green to a similar extent as the wild type, whereas the greening was significantly reduced under low light conditions. During greening, high light irradiation increased the level of PORC protein, and the greening of porC was repressed under high light conditions. The porB, but not porC, etiolated seedling was more sensitive to the far-red block of greening than the wild type, which is caused by depletion of endogenous POR proteins resulting in photo-oxidative damage. These results suggest that, at the onset of greening, PLBs are important for efficient capture of light energy for photoconversion under various light conditions, and PORC, which is induced by high light irradiation, contributes to photoprotection during greening of the etiolated seedlings.     

Multiplicity of different cell- and organ-specific import routes for the NADPH-protochlorophyllide oxidoreductases A and B in plastids of Arabidopsis seedlings

The NADPH-dependent protochlorophyllide (Pchlide) oxidoreductase (POR) is a photoenzyme that requires light for its catalytic activity and uses Pchlide itself as a photoreceptor. In Arabidopsis there are three PORs denoted PORA, PORB and PORC. The PORA and PORB genes are strongly expressed early in seedling development. In contrast to PORB the import of PORA into plastids of cotyledons is substrate-dependent and organ-specific. These differences in the import reactions between PORA and PORB most likely are due to different import mechanisms that are responsible for the uptake of these proteins. The two major core constituents of the translocon of the outer plastid envelope, Toc159 and Toc34, have been implicated in the binding and recognition of precursors of nuclear-encoded plastid proteins. Their involvement in conferring substrate dependency and organ specificity of PORA import was analyzed in intact Arabidopsis seedlings of wild type and the three mutants ppi3, ppi1 and ppi2 that are deficient in atToc34, atToc33, a closely related isoform of atToc34, and atToc159. Whereas none of these three Toc constituents is required for maintaining the organ specificity and substrate dependency of PORA import, atToc33 is indispensable for the import of PORB in cotyledons and true leaves suggesting that in these parts of the plant translocation of PORA and PORB occurs via two distinct import pathways. The analysis of PORA and PORB import into plastids of intact seedlings revealed an unexpected multiplicity of import routes that differed by their substrate, cell, tissue and organ specificities. This versatility of pathways for protein targeting to plastids suggests that in intact seedlings not only the constituents of the core complex of import channels but also other factors are involved in mediating the import of nuclear-encoded plastid proteins.     

Protochlorophyllide-independent import of two NADPH:Pchlide oxidoreductase proteins (PORA and PORB) from barley into isolated plastids

The enzyme catalysing the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), NADPH:Pchlide oxidoreductase (POR; EC 1.6.99.1), is a nuclear-encoded protein that is post-translationally imported to the plastid. In barley and Arabidopsis thaliana , the reduction of Pchlide is controlled by two different PORs, PORA and PORB. To characterise the possible Pchlide dependency for the import reaction, radiolabelled precursor proteins of barley PORA and PORB (pPORA and pPORB, respectively) were used for in vitro assays with isolated plastids of barley and pea with different contents of Pchlide. To obtain plastids with different endogenous levels of Pchlide, several methods were used. Barley plants were grown in darkness or in greenhouse conditions for 6 days. Alternatively, greenhouse-grown pea plants were incubated for 4 days in darkness before plastid isolation, or chloroplasts isolated from greenhouse-grown plants were incubated with Δ -aminolevulinic acid (ALA), an early precursor in the Chl biosynthesis resulting in elevated Pchlide contents in the plastids. Both barley pPORA and pPORB were effectively imported into barley and pea chloroplasts isolated from the differentially treated plants, including those isolated from greenhouse-grown plants. The absence or presence of Pchlide did not significantly affect the import capacity of barley pPORA or pPORB. Assays performed on stroma-enriched fractions from chloroplasts and etioplasts of barley indicated that no post-import degradation of the proteins occurred in the stroma, irrespective of whether the incubation was performed in darkness or in light.     

Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants

Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB , that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F₆₅₅, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F₆₅₅.     

NADPH:protochlorophyllide oxidoreductase B (PORB) action in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> revisited through transgenic expression of engineered barley PORB mutant proteins

NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme for the light-induced greening of etiolated angiosperm plants. It belongs to the ‘RED’ family of reductases, epimerases and dehydrogenases. All POR proteins characterized so far contain evolutionarily conserved cysteine residues implicated in protochlorophyllide (Pchlide)-binding and catalysis. cDNAs were constructed by site-directed mutagenesis that encode PORB mutant proteins with defined Cys→Ala exchanges. These cDNAs were expressed in transgenic plants of a PORB-deficient knock-out mutant (porB) of Arabidopsis thaliana. Results show that porB plants expressing PORB mutant proteins with Ala substitutions of Cys276 or Cys303 are hypersensitive to high-light conditions during greening. Hereby, failure to assemble higher molecular weight complexes of PORB with its twin isoenzyme, PORA, as encountered with (Cys303→Ala)-PORB plants, caused more severe effects than replacing Cys276 by an Ala residue in the active site of the enzyme, as encountered in (Cys276→Ala)-PORB plants. Our results are consistent with the presence of two distinct pigment binding sites in PORB, with Cys276 establishing the active site of the enzyme and Cys303 providing a second, low affinity pigment binding site that is essential for the assembly of higher molecular mass light-harvesting PORB::PORA complexes and photoprotection of etiolated seedlings. Failure to assemble such complexes provoked photodynamic damage through the generation of singlet oxygen. Together, our data highlight the importance of PORB for Pchlide homoeostasis and greening in Arabidopsis.     

POR hits the road: import and assembly of a plastid protein

The biosynthesis of chlorophyll is a strictly light-dependent multistep process in higher plants. The light-dependent step is catalysed by NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1), which reduces protochlorophyllide (Pchlide) to chlorophyllide (Chlide). POR is nucleus-encoded and post-translationally imported into plastids. It has been proposed that the import of a POR protein isozyme (PORA) is totally dependent on Pchlide and uses a novel import pathway. This proposal is based on findings that PORA import only occurs in the presence of Pchlide and that the presence of overexpressed precursor of Rubisco small subunit (pSS), a protein which is known to use the general import pathway, does not outcompete PORA import. Another study demonstrated that POR precursor protein (pPOR) can be cross-linked to one of the components in the translocation machinery, Toc75, in the absence of Pchlide, and that its import can be outcompeted by the addition of the pSS. This indicates that pSS and pPOR may use the same translocation mechanism. Thus, POR does not necessarily need Pchlide for import – which is in contrast to earlier observations – and the exact POR import mechanism remains unresolved. Once in the stroma, the POR transit peptide is cleaved off and the mature POR protein is associated to the plastid inner membranes. Formation of the correct membrane–associated, thermolysin-protected assembly is strictly dependent of NADPH. As a final step, the formation of the NADPH-Pchlide-POR complex occurs. When POR accumulates in the membranes of proplastids, an attraction of monogalactosyl diacylglycerol (MGDG) can occur, leading to the formation of prolamellar bodies (PLBs) and the development of etioplasts in darkness.     

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.     

Assembly of NADPH:protochlorophyllide oxidoreductase complex is needed for effective greening of barley seedlings

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the key enzyme in the light-induced greening of higher plants. A unique light-harvesting POR:Pchlide complexes (LHPP) has been found in barley etioplasts, but not in other plant species. Why PORs from barley, but not from other plants, can form LHPP? And its function is not well understood. We modeled the barley and Arabidopsis POR proteins and compared molecular surface. The results confirm the idea that barley PORA can form a five-unit oligomer that interacts with a single PORB. Chemical treatment experiments indicated that POR complex may be formed by dithiol oxidation of cysteines of two adjacent proteins. We further showed that LHPP assembly was needed for barley POR functions and seedling greening. On the contrary, Arabidopsis POR proteins only formed dimers, which were not related to the functions or the greening. Finally, POR complex assembly (including LHPP and POR dimers) did not affect the formation of prolamellar bodies (PLBs) that function for efficient capture of light energy for photo conversion in etioplasts.     

Regulation of etioplast pigment-protein complexes, inner membrane architecture, and protochlorophyllide a chemical heterogeneity by light-dependent NADPH:protochlorophyllide oxidoreductases A and B

The etioplast of dark-grown angiosperms is characterized by the prolamellar body (PLB) inner membrane, the absence of chlorophyll, and the accumulation of divinyl and monovinyl derivatives of protochlorophyll(ide) a [Pchl(ide) a]. Either of two structurally related, but differentially expressed light-dependent NADPH:Pchlide oxidoreductases (PORs), PORA and PORB, can assemble the PLB and form dark-stable ternary complexes containing enzymatically photoactive Pchlide-F655. Here we have examined in detail whether these polypeptides play redundant roles in etioplast differentiation by manipulating the total POR content and the PORA-to-PORB ratio of etiolated Arabidopsis seedlings using antisense and overexpression approaches. POR content correlates closely with PLB formation, the amounts, spectroscopic properties, and photoreduction kinetics of photoactive Pchlide, the ratio of photoactive Pchlide-F655 to non-photoactive Pchl(ide)-F632, and the ratio of divinyl- to monovinyl-Pchl(ide). This last result defines POR as the first endogenous protein factor demonstrated to influence the chemical heterogeneity of Pchl(ide) in angiosperms. It is intriguing that excitation energy transfer between different spectroscopic forms of Pchl(ide) in etiolated cotyledons remains largely independent of POR content. We therefore propose that the PLB contains a minimal structural unit with defined pigment stoichiometries, within which a small amount of non-photoactive Pchl(ide) transfers excitation energy to a large excess of photoactive Pchlide-F655. In addition, our data suggests that POR may bind not only stoichiometric amounts of photoactive Pchlide, but also substoichiometric amounts of non-photoactive Pchl(ide). We conclude that the typical characteristics of etioplasts are closely related to total POR content, but not obviously to the specific presence of PORA or PORB.     

POR - import and membrane association of a key element in chloroplast development

The development of proplastids or etioplasts to chloroplast is visualized by the accumulation of chlorophyll in leaves of higher plants. The biosynthesis of chlorophyll includes a light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This light-dependent step is catalysed by the nucleus-encoded NADPH:Pchlide oxidoreductase (POR, EC 1.6.99.1). POR is active within plastids and therefore has to be translocated over the plastid envelope membranes. The import of chloroplast proteins seems to follow a general import pathway using translocons at the outer and inner envelope membrane. POR cross-linking to Toc75, one of the major translocon components at the outer envelope membrane, indicates its use of the general import pathway. However, since variations exist within the so-called general import pathway one has to consider previous data suggesting a novel totally Pchlide-dependent import pathway of one POR isoform, PORA. The suggested Pchlide dependency of POR import is discussed since recent observations contradict this idea. In the stroma the POR transit peptide is cleaved off and the mature POR protein is targeted to the plastid inner membranes. The correct and stable association of POR to the membrane requires the cofactor NADPH. Functional activity of POR calls for formation of an NADPH–Pchlide–POR complex, a formation that probably takes place after the membrane association and is dependent on a phosphorylation reaction.     

Two NADPH:Protochlorophyllide Oxidoreductases in Barley: Evidence for the Selective Disappearance of PORA during the Light-Induced Greening of Etiolated Seedlings

Chlorophyll synthesis in barley is controlled by two different light-dependent NADPH:protochlorophyllide oxidoreductases, termed PORA and PORB. PORA is present abundantly in etioplasts but selectively disappears soon after the beginning of illumination. This negative light effect is mediated simultaneously at three different levels. First, the concentration of porA mRNA declines drastically during illumination of dark-grown seedlings. Second, the plastids' ability to import the precursor of PORA (pPORA) is reduced during the transition from etioplasts to chloroplasts. This effect is due to a rapid decline in the plastidic level of protochlorophyllide (Pchlide), which is required for the translocation of the pPORA. Third, PORA becomes selectively destabilized in illuminated seedlings. When illuminated, PORA-Pchlide-NADPH complexes formed in the dark photoreduce their Pchlide to Chlide and become simultaneously susceptible to attack by plastid proteases. The PORA-degrading protease activity is not detectable in etioplasts but is induced during illumination. In contrast to PORA, the second Pchlide-reducing enzyme, PORB, remains operative in both illuminated and green plants. Its translocation into plastids does not depend on its substrate, Pchlide.     

Role of Arabidopsis CHL27 protein for photosynthesis, chloroplast development and gene expression profiling

In Chl biosynthesis, aerobic Mg-protoporphyrin IX monomethyl ester (MPE) cyclase is a key enzyme involved in the synthesis of protochlorophyllide a, and its membrane-bound component is known to be encoded by homologs of CHL27 in photosynthetic bacteria, green algae and plants. Here, we report that the Arabidopsis chl27-t knock-down mutant exhibits retarded growth and chloroplast developmental defects that are caused by damage to PSII reaction centers. The mutant contains a T-DNA insertion within the CHL27 promoter that dramatically reduces the CHL27 mRNA level. chl27-t mutant plants grew slowly with a pale green appearance, suggesting that they are defective in Chl biosynthesis. Chl fluorescence analysis showed significantly low photosynthetic activity in chl27-t mutants, indicating damage in their PSII reaction centers. The chl27-t mutation also conferred severe defects in chloroplast development, including the unstacking of thylakoid membranes. Microarray analysis of the chl27-t mutant showed repression of numerous nuclear genes involved in photosynthesis, including those encoding components of light-harvesting complex I (LHCI) and LHCII, and PSI and PSII, which accounts for the defects in photosynthetic activity and chloroplast development. In addition, the microarray data also revealed the significant repression of genes such as PORA and AtFRO6 for Chl biosynthesis and iron acquisition, respectively, and, furthermore, implied that there is cross-talk in the Chl biosynthetic pathway among the PORA, AtFRO6 and CHL27 proteins.     

Chlorophyll Synthesis in a Deetiolated (det340) Mutant of Arabidopsis without NADPH-Protochlorophyllide (PChlide) Oxidoreductase (POR) A and Photoactive PChlide-F655

Chlorophyll (Chl) synthesis in Arabidopsis is controlled by two light-dependent NADPH-protochlorophyllide (PChlide) oxidoreductases (PORs), one (POR A) that is active transiently in etiolated seedlings at the beginning of illumination and another (POR B) that also operates in green plants. The function of these two enzymes during the light-induced greening of dark-grown seedlings has been studied in the wild type and a deetiolated (det340) mutant of Arabidopsis. One of the consequences of the det mutation is that POR A is constitutively down-regulated, and therefore, synthesis of the POR A enzyme is shut off. When grown in the dark, the det340 mutant lacks POR A and the photoactive PChlide-F655 species but maintains the second PChlide reductase, POR B. Previously, photoactive PChlide-F655 has often been considered to be the only PChlide form that leads to Chl formation. Despite its deficiency in POR A and photoactive PChlide-F655, the det340 mutant is able to green when placed in the light. Chl accumulation, however, proceeds abnormally. At the beginning of illumination, seedlings of det340 mutants are extremely susceptible to photooxidative damage and accumulate Chl only at extremely low light intensities. They form core complexes of photosystems I and II but are almost completely devoid of light-harvesting structures. The results of this study demonstrate that in addition to the route of Chl synthesis that has been studied extensively in illuminated dark-grown wild-type plants, a second branch of Chl synthesis exists that is driven by POR B and does not require POR A.