Identification of Genes Associated with Chlorophyll Accumulation in Flower Petals

Plants have an ability to prevent chlorophyll accumulation, which would mask the bright flower color, in their petals. In contrast, leaves contain substantial amounts of chlorophyll, as it is essential for photosynthesis. The mechanisms of organ-specific chlorophyll accumulation are unknown. To identify factors that determine the chlorophyll content in petals, we compared the expression of genes related to chlorophyll metabolism in different stages of non-green (red and white) petals (very low chlorophyll content), pale-green petals (low chlorophyll content), and leaves (high chlorophyll content) of carnation (Dianthus caryophyllus L.). The expression of many genes encoding chlorophyll biosynthesis enzymes, in particular Mg-chelatase, was lower in non-green petals than in leaves. Non-green petals also showed higher expression of genes involved in chlorophyll degradation, including STAY-GREEN gene and pheophytinase. These data suggest that the absence of chlorophylls in carnation petals may be caused by the low rate of chlorophyll biosynthesis and high rate of degradation. Similar results were obtained by the analysis of Arabidopsis microarray data. In carnation, most genes related to chlorophyll biosynthesis were expressed at similar levels in pale-green petals and leaves, whereas the expression of chlorophyll catabolic genes was higher in pale-green petals than in leaves. Therefore, we hypothesize that the difference in chlorophyll content between non-green and pale-green petals is due to different levels of chlorophyll biosynthesis. Our study provides a basis for future molecular and genetic studies on organ-specific chlorophyll accumulation.     

Regreening of senescent Nicotiana leaves. I. Reappearance of NADPH-protochlorophyllide oxidoreductase and light-harvesting chlorophyll a/b-binding protein

Decapitation of Nicotiana rustica L. plants above a single senescent leaf induced regreening, which was promoted by cytokinin treatment. Regreening required low light. The decline in leaf protein content and increase in protease activity seen during senescence were reversed on regreening. Western blotting showed that light-harvesting chlorophyll a/b-binding protein declined considerably during senescence, but on regreening it increased back to the levels seen in green leaves. NADPH-protochlorophyllide oxidoreductase (POR) was found by Western blotting at high levels in etiolated cotyledons, but at low levels in green leaves and not at all in senescent leaves. However, POR reappeared in regreening leaves, and cytokinin accelerated its increase.     

Novel Insights into the Enzymology,Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms

The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently, angiosperms become etiolated in the absence of light, since the reduction of Pchlide in angiosperms is solely dependent on POR. In eukaryotic phototrophs, POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids. POR possesses unique features, its light-dependent catalytic activity, accumulation in plastids of dark-grown angiosperms (etioplasts) via binding to its substrate, Pchlide, and cofactor, NADPH, resulting in the formation of prolamellar bodies (PLBs), and rapid degradation after catalysis under subsequent illumination. During the last decade, considerable progress has been made in the study of the gene organization, catalytic mechanism, membrane association, regulation of the gene expression, and physiological function of POR. In this review, we provide a brief overview of DPOR and then summarize the current state of knowledge on the biochemistry and molecular biology of POR mainly in angiosperms. The physiological and evolutional implications of POR are also discussed.     

Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.)

The effects of leaf developmental age on the expression of three nuclear gene families in pea (Pisum sativum L.) coding for enzymes of chlorophyll and heme biosynthesis have been examined. The steady-state levels of mRNAs encoding aminolevulinic acid (ALA) dehydratase, porphobilinogen (PBG) deaminase, and NADPH:protochlorophyllide reductase were measured by RNA gel blot and quantitative slot-blot analyses in the foliar leaves of embryos that had imbibed for 12 to 18 h and leaves of developing seedlings grown either in total darkness or under continuous white light for up to 14 d after imbibition. Both ALA dehydratase and PBG deaminase mRNAs were detectable in embryonic leaves, whereas mRNA encoding the NADPH:protochlorophyllide reductase was not observed at this early developmental stage. All three gene products were found to increase to approximately the same extent in the primary leaves of pea seedlings during the first 6 to 8 d after imbibition (postgermination) regardless of whether the plants were grown in darkness or under continuous white-light illumination. In the leaves of dark-grown seedlings, the highest levels of message accumulation were observed at approximately 8 to 10 d postgermination, and, thereafter, a steady decline in mRNA levels was observed. In the leaves of light-grown seedlings, steady-state levels of mRNA encoding the three chlorophyll biosynthetic enzymes were inversely correlated with leaf age, with youngest, rapidly expanding leaves containing the highest message levels. A corresponding increase in the three enzyme protein levels was also found during the early stages of development in the light or darkness; however, maximal accumulation of protein was delayed relative to peak levels of mRNA accumulation. We also found that although protochlorophyllide was detectable in the leaves immediately after imbibition, the time course of accumulation of the phototransformable form of the molecule coincided with NADPH:protochlorophyllide reductase expression. In studies in which dark-grown seedlings of various ages were subsequently transferred to light for 24 and 48 h, the effect of light on changes in steady-state mRNA levels was found to be more pronounced at later developmental stages. These results suggest that the expression of these three genes and likely those genes encoding other chlorophyll biosynthetic pathway enzymes are under the control of a common regulatory mechanism. Furthermore, it appears that not light, but rather as yet unidentified endogenous factors, are the primary regulatory factors controlling gene expression early in leaf development.     

Pigment variation and transcriptional response of the pigment synthesis pathway in the S2309 triple-color ornamental kale (Brassica oleracea L. var. acephala) line

Ornamental kale is popular because of its colorful leaves and few studies have investigated the mechanism of color changes. In this study, an ornamental kale line (S2309) with three leaf colors was developed. Analysis of the anthocyanin, chlorophyll, and carotenoid contents and RNA-seq were performed on the three leaf color types. There was less chlorophyll in the white leaves and purple leaves than in the green leaves, and the anthocyanin content was greatest in the purple leaves. All the downregulated DEGs related to chlorophyll metabolism were detected only in the S2309_G vs. S2309_W comparison, which indicated that the decrease in chlorophyll content was caused mainly by the inhibition of chlorophyll biosynthesis during the leaf color change from green to white. Moreover, the expression of 19 DEGs involved in the anthocyanin biosynthesis pathway was upregulated. These results provide new insight into the mechanisms underlying the three-color formation.     

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.     

Defective etioplasts observed in variegation mutants may reveal the light-independent regulation of white/yellow sectors of Arabidopsis leaves

Leaf variegation resulting from nuclear gene mutations has been used as a model system to elucidate the molecular mechanisms of chloroplast development. Since most variegation genes also function in photosynthesis, it remains unknown whether their roles in photosynthesis and chloroplast development are distinct. Here, using the variegation mutant thylakoid formation1 (thf1) we show that variegation formation is light independent. It was found that slow and uneven chloroplast development in thf1 can be attributed to defects in etioplast development in darkness. Ultrastructural analysis showed the coexistence of plastids with or without prolamellar bodies (PLB) in cells of thf1, but not of WT. Although THF1 mutation leads to significant decreases in the levels of Pchlide and Pchllide oxidoreductase (POR) expression, genetic and 5-aminolevulinic acid (ALA)-feeding analysis did not reveal Pchlide or POR to be critical factors for etioplast formation in thf1. Northern blot analysis showed that plastid gene expression is dramatically reduced in thf1 compared with that in WT, particularly in the dark. Our results also indicate that chlorophyll biosynthesis and expression of plastidic genes are coordinately suppressed in thf1. Based on these results, we propose a model to explain leaf variegation formation from the plastid development perspective.     

Arabidopsis miR171-Targeted Scarecrow-Like Proteins Bind to GT cis-Elements and Mediate Gibberellin-Regulated Chlorophyll Biosynthesis under Light Conditions

An extraordinarily precise regulation of chlorophyll biosynthesis is essential for plant growth and development. However, our knowledge on the complex regulatory mechanisms of chlorophyll biosynthesis is very limited. Previous studies have demonstrated that miR171-targeted scarecrow-like proteins (SCL6/22/27) negatively regulate chlorophyll biosynthesis via an unknown mechanism. Here we showed that SCLs inhibit the expression of the key gene encoding protochlorophyllide oxidoreductase (POR) in light-grown plants, but have no significant effect on protochlorophyllide biosynthesis in etiolated seedlings. Histochemical analysis of β-glucuronidase (GUS) activity in transgenic plants expressing pSCL27::rSCL27-GUS revealed that SCL27-GUS accumulates at high levels and suppresses chlorophyll biosynthesis at the leaf basal proliferation region during leaf development. Transient gene expression assays showed that the promoter activity of PORC is indeed regulated by SCL27. Consistently, chromatin immunoprecipitation and quantitative PCR assays showed that SCL27 binds to the promoter region of PORC in vivo. An electrophoretic mobility shift assay revealed that SCL27 is directly interacted with G(A/G)(A/T)AA(A/T)GT cis-elements of the PORC promoter. Furthermore, genetic analysis showed that gibberellin (GA)-regulated chlorophyll biosynthesis is mediated, at least in part, by SCLs. We demonstrated that SCL27 interacts with DELLA proteins in vitro and in vivo by yeast-two-hybrid and coimmunoprecipitation analysis and found that their interaction reduces the binding activity of SCL27 to the PORC promoter. Additionally, we showed that SCL27 activates MIR171 gene expression, forming a feedback regulatory loop. Taken together, our data suggest that the miR171-SCL module is critical for mediating GA-DELLA signaling in the coordinate regulation of chlorophyll biosynthesis and leaf growth in light.     

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 regulation of NADPH-protochlorophyllide oxidoreductases A and B in green barley plants kept under a diurnal light/dark cycle

In etiolated barley (Hordeum vulgare L.) seedlings the light-induced accumulation of chlorophyll is controlled by two light-dependent NADPH-proto-chlorophyllide oxidoreductase (POR; EC 1.6.99.1) enzymes. While the concentration of one of these enzymes (POR A) and its mRNA rapidly decline during illumination, the second POR protein (POR B) and its mRNA remain at an approximately constant level during the transition from dark growth to the light. These results may suggest that only one of the enzymes, POR B, operates throughout the greening process and in light-adapted mature plants while the second enzyme, POR A, is active only in etiolated seedlings at the beginning of illumination. The fate of the two POR proteins and their mRNAs in fully green plants, however, has not been studied yet. In the present work we determined changes in the level of POR A and POR B proteins and mRNAs in green barley plants kept under a diurnal 12 h light/12 h dark cycle. In green barley plants, not only POR B is present but also trace amounts of POR A continue to reappear transiently at the end of a night period and seem to be involved in the synthesis and accumulation of chlorophyll at the beginning of each day.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Lhcb light-harvesting chlorophyll a/b protein - Pchlide protochlorophyllide - POR NADPH-protochlorophyllide oxidoreductase Dedicated to Horst Senger on the occasion of his 65th birthday.We thank Dr. Dieter Rubli for photography and Renate Langjahr for typing. This work was supported by the Swiss National Science Foundation and the ETH-Zürich.     

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.     

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.     

Effect of transient accumulation of anthocyanin on leaf development and photoprotection of Fagopyrum dibotrys mutant

Changes in pigments contents, leaf area, leaf dry mass per unit area (LMA), photosynthetic rate and chlorophyll a fluorescence were investigated in developing leaves of Fagopyrum dibotrys Hara. mutant. Anthocyanins transiently accumulate below the upper epidermis during leaf ontogeny of this mutant. Red leaves possessed lower Chl content, LMA, photosynthetic rate and apparent carboxylation efficiency than green leaves. However, content of anthocyanins declined and above mentioned parameters increased during further leaf development. In both red and green leaves, chronic photoinhibition did not take place according to variable to maximum chlorophyll fluorescence ratio (F_v/F_m). Red leaves had higher non-photochemical quenching (NPQ) and higher PS 2 efficiency.     

Functional role of red (retro)-carotenoids as passive light filters in the leaves of Buxus sempervirens L.: increased protection of photosynthetic tissues?

Red (retro)-carotenoids accumulate in chloroplasts of Buxus sempervirens leaves during the process of winter leaf acclimation. As a result of their irregular presence, different leaf colour phenotypes can be found simultaneously in the same location. Five different colour phenotypes (green, brown, red, orange, and yellow), with a distinct pattern of pigment distribution and concentration, have been characterized. Leaf reddening due to the presence of anthocyanins or carotenoids, is a process frequently observed in plant species under photoinhibitory situations. Two main hypotheses have been proposed to explain the function of such colour change: antioxidative protection exerted by red-coloured molecules, and green light filtering. The potential photoprotective role of red (retro-) carotenoids as light filters was tested in Buxus sempervirens leaves. In shade leaves of this species the upper (adaxial) mesophyll of the lamina was replaced by the equivalent upper part of a different colour phenotype. These hybrid leaves were exposed to a photoinhibitory treatment in order to compare the photoprotective effect exerted by adaxial parts of phenotypes with a different proportion of red (retro)-carotenoids in the lower mesophyll of a shade leaf. The results indicated that the presence of red (retro)-carotenoids in the upper mesophyll did not increase photoprotection of the lower mesophyll when compared with chlorophyll, and the best protection was achieved by an upper green layer. This was due to the fact that the extent of photoinhibition was proportional to the amount of red light transmitted by the upper mesophyll and/or to the chlorophyll pool located above. These results do not exclude a protective function of carotenoids in the upper leaf layer, but imply that, at least under the conditions of this experiment, the accumulation of red pigments in the outer leaf layers does not increase photoprotection in the lower mesophyll.