Analysis of protochlorophyllide reaccumulation in the phytochrome chromophore-deficient aurea and yg-2 mutants of tomato by in vivo fluorescence spectroscopy

The aurea and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum) are unable to synthesize the phytochrome chromophore from heme resulting in a block of this branch of the tetrapyrrole pathway. We have previously shown that these mutants also exhibit an inhibition of protochlorophyllide (Pchlide) synthesis and it has been hypothesised that this is due to feedback inhibition by heme on the synthesis of 5-aminolevulinic acid (ALA). In this study we have investigated Pchlide reaccumulation in cotyledons from etiolated wild-type (WT), aurea and yg-2 seedlings using low-temperature fluorescence spectroscopy. WT cotyledons showed two characteristic Pchlide emission maxima at 630 nm (F630) and 655 nm (F655) respectively, while the aurea and yg-2 mutants contained only phototransformable Pchlide F655. Following a white-light flash to WT cotyledons, reaccumulation of phototransformable Pchlide F655 in the first 30 min was absolutely dependent on the presence of Pchlide F630 before the flash. Reaccumulation of Pchlide F630 was not apparent until at least 2 h after the phototransformation. In contrast, Pchlide F630 never accumulated in aurea cotyledons. The relative rates of both Pchlide F655 and total Pchlide synthesis were approximately twice as high in WT compared to aurea. Measurement of ALA synthesis capacity during this period showed that the reduced rate of Pchlide reaccumulation in aurea was due to an inhibition at this step of the pathway. In addition, feeding of ALA resulted in a substantial and equal increase of non-phototransformable Pchlide in both WT and aurea indicating that aurea cotyledons are capable of accumulating high levels of Pchlide that is not associated to the active site of NADPH:Pchlide oxidoreductase (POR). The implications of these results for the mechanism of inhibition of Pchlide synthesis in phytochrome chromophore-deficient mutants and the role of non-phototransformable Pchlide F630 during plastid development are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Altered etioplast development in phytochrome chromophore-deficient mutants

Inhibition of chromophore synthesis in the phytochrome-deficient aurea (au) and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum L.) results in a severe reduction of protochlorophyllide (Pchlide) accumulation in dark-grown hypocotyls. Experiments with apophytochrome-deficient mutants indicate that the inhibition of Pchlide accumulation results from two separate effects: one dependent on the activity of phytochromes A and B1 and one phytochrome-independent effect that is attributed to a feedback inhibition of the tetrapyrrole biosynthesis pathway. Cotyledons only show phytochrome-independent inhibition of Pchlide synthesis. Analysis of NADPH:protochlorophyllide oxidoreductase levels by western blotting showed that the reduction in Pchlide in au and yg-2 is accompanied by a correlative, but less substantial, decrease in NADPH:protochlorophyllide oxidoreductase. Consistent with this result, in vivo fluorescence spectra demonstrate that both mutants are primarily deficient in non-phototransformable Pchlide. Analysis of etioplast structure indicates that plastid development in au and yg-2 is retarded in hypocotyls and partially impaired in cotyledons, again correlating with the reduction in Pchlide. Since Pchlide synthesis is also reduced in chromophore-deficient mutants of pea (Pisum sativum L.) and Arabidopsis thaliana (L.) Heynh. (Landsberg erecta) these results may be significant for explaining aspects of the phenotype of this mutant class that are independent of the loss of phytochrome.     

Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts : III. Evidence for Functional Heterogeneity of the ALA Pool

Gabaculine and 4-amino-5-hexynoic acid (AHA) up to 3.0 millimolar concentration strongly inhibited 5-aminolevulinic acid (ALA) synthesis in developing cucumber (Cucumis sativus L. var Beit Alpha) chloroplasts, while they hardly affected protochlorophyllide (Pchlide) synthesis. Exogenous protoheme up to 1.0 micromolar had a similar effect. Exogenous glutathione also exhibited a strong inhibitory effect on ALA synthesis in organello but hardly inhibited Pchlide synthesis. Pchlide synthesis in organello was highly sensitive to inhibition by levulinic acid, both in the presence and in the absence of gabaculine, indicating that the Pchlide was indeed formed from precursor(s) before the ALA dehydratase step. The synthesis of Pchlide in the presence of saturating concentrations of glutamate was stimulated by exogenous ALA, confirming that Pchlide synthesis was limited at the formation of ALA. The gabaculine inhibition of ALA accumulation occurred whether levulinic acid or 4,6-dioxohepatonic acid was used in the ALA assay system. ALA overproduction was also observed in the absence of added glutamate and was noticeable after 10-minute incubation. These observations suggest that although Pchlide synthesis in organello is limited by ALA formation, it does not utilize all the ALA that is made in the in organello assay system. Gabaculine, AHA, and probably also protoheme, inhibit preferentially the formation of that portion of ALA that is not destined for Pchlide. A model proposing a heterogenous ALA pool is described.     

Concurrent interactions of heme and FLU with Glu tRNA reductase (HEMA1), the target of metabolic feedback inhibition of tetrapyrrole biosynthesis, in dark- and light-grown Arabidopsis plants

The regulation of tetrapyrrole biosynthesis in higher plants has been attributed to metabolic feedback inhibition of Glu tRNA reductase by heme. Recently, another negative regulator of tetrapyrrole biosynthesis has been discovered, the FLU protein. During an extensive second site screen of mutagenized flu seedlings a suppressor of flu, ulf3, was identified that is allelic to hy1 and encodes a heme oxygenase. Increased levels of heme in the hy1 mutant have been implicated with inhibiting Glu tRNA reductase and suppressing the synthesis of delta-aminolevulinic acid (ALA) and Pchlide accumulation. When combined with hy1 or ulf3 upregulation of ALA synthesis and overaccumulation of protochlorophyllide in the flu mutants were severely suppressed supporting the notion that heme antagonizes the effect of the flu mutation by inhibiting Glu tRNA reductase independently of FLU. The coiled-coil domain at the C-terminal end of Glu tRNA reductase interacts with FLU, whereas the N-terminal site of Glu tRNA reductase that is necessary for the inhibition of the enzyme by heme is not required for this interaction. The interaction with FLU is specific for the Glu tRNA reductase encoded by HEMA1 that is expressed in photosynthetically active tissues. FLU seems to be part of a second regulatory circuit that controls chlorophyll biosynthesis by interacting directly with Glu tRNA reductase not only in etiolated seedlings but also in light-adapted green plants.     

Overexpression of HEMA1 encoding glutamyl-tRNA reductase

5-Aminolevulinic acid (ALA) synthesis has been shown to be the rate limiting step of tetrapyrrole biosynthesis. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of plant ALA synthesis and is controlled by interacting regulators, such as heme and the FLU protein. Induced inactivation of the HEMA1 gene encoding GluTR by RNAi expression in tobacco resulted in a reduced activity of Mg chelatase and Fe chelatase indicating a feed-forward regulatory mechanism that links ALA synthesis posttranslationally with late enzymes of tetrapyrrole biosynthesis (Hedtke et al., 2007). Here, the regulatory impact of GluTR was investigated by overexpression of AtHEMA1 in Arabidopsis and tobacco plants. Light-dependent ALA synthesis cannot benefit from an up to 7-fold induced expression of GluTR in Arabidopsis. While constitutive AtHEMA1 overexpression in tobacco stimulates ALA synthesis by 50-90% during light-exposed growth of seedlings, no increase in heme and chlorophyll contents is observed. HEMA1 overexpression in etiolated and dark-grown Arabidopsis and tobacco seedlings leads to additional accumulation of protochlorophyllide. As excessive accumulation of GluTR does not correlate with increased ALA formation, it is hypothesized that ALA synthesis is additionally limited by other effectors that balance the allocation of ALA with the activity of enzymes of chlorophyll and heme biosynthesis.     

Analysis of phytochrome-deficient yellow-green-2 and aurea mutants of tomato

Two alleles of the yellow-green-2 ( yg-2) and eight different alleles of the aurea ( au ) locus of tomato ( Lycopersicon esculentum Mill.) were compared. All are characterized by a paler green colour compared with wild-type (WT), an elongated hypocotyl in red light, and low or below detection limits of spectrophotometrically active phytochrome. Hypocotyl length was variable in white light, ranging from that of WT to more elongated. Immunochemical analysis revealed that etiolated seedlings of the yg-2 mutant have approximately 25% of the WT level of phytochrome A protein (PHYA), whereas that of phytochrome B protein (PHYB) is normal. In this it resembles the au mutant. The au,yg-2 double mutant has a more extreme chlorophyll deficiency than either parent. Since the yg-2 and au mutants have a less severe phenotype at the adult stage, that is, are leaky, the additive effect can be explained by assuming that the mutants control two steps in the chromophore biosynthesis pathway. Combination, by crossing, of the yg-2 and au mutants with a transgenic tomato line that overexpresses oat phytochrome A3 (PhyA-3) essentially failed to restore the WT phenotype under white fluorescent light conditions, although under greenhouse conditions some evidence for increased sensitivity to light was observed. Immunochemically, oat PHYA-3 protein is detectable in both the yg-2,PhyA-3 and au,PhyA-3 'double' mutants. Spectrophotometrical analysis, however, revealed that holophytochrome was undetectable in the yg-2,PhyA-3 and au,PhyA-3 'double' mutants. These results are compatible with both mutants being disturbed in phytochrome chromophore biosynthesis.     

Inhibition of phytochrome synthesis by gabaculine

Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid), a transaminase inhibitor, also inhibits chlorophyll formation in plants, and the effect of this compound can be counteracted by 5-aminolevulinic acid (ALA) (Flint, personal communication, 1984). Since it is probable that ALA also serves as a precursor to phytochrome, the effects of gabaculine on phytochrome synthesis in developing etiolated seedlings were examined using in vivo spectrophotometry. Preemergence treatment with gabaculine was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis, perhaps due to preexisting phytochrome in the seed. Foliar treatment of etiolated pea seedlings prior to light-induced destruction of phytochrome inhibited subsequent phytochrome resynthesis in the dark. These results suggest that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein.     

Expression of a Soybean Gene Encoding the Tetrapyrrole-Synthesis Enzyme Glutamyl-tRNA Reductase in Symbiotic Root Nodules

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor δ-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3′ portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.Soybean (Glycine max) and numerous other legumes can establish a symbiosis with rhizobia, resulting in the formation of root nodules comprising specialized plant and bacterial cells (for review, see Mylona et al., 1995). Rhizobia reduce atmospheric nitrogen to ammonia within nodules, which is assimilated by the plant host to fulfill its nutritional nitrogen requirement. The high energy requirement for nitrogen fixation necessitates efficient respiration by the prokaryote within the microaerobic milieu of the nodule. The plant host synthesizes a nodule-specific hemoglobin (leghemoglobin) that serves to facilitate oxygen diffusion to the bacterial endosymbiont and to buffer the free oxygen concentration at a low tension (for review, see Appleby, 1992). Both of these functions require that the hemoglobin concentration be high, and, indeed, it exceeds 1 mm in soybean nodules (Appleby, 1984) and is the predominant plant protein in that organ. Once thought to be confined to legume nodules, hemoglobins are found throughout the plant kingdom, and leghemoglobin likely represents a specialization of a general plant phenomenon (for review, see Hardison, 1996). A gene encoding a nonsymbiotic hemoglobin has been identified in soybean and other legumes (Andersson et al., 1996); therefore, expression in nodules involves the specific activation of a subset of genes within a gene family. Leghemoglobin genes may have arisen from gene duplication, followed by specialization (Andersson et al., 1996).Hemes and chlorophyll are tetrapyrroles synthesized from common precursors; chlorophyll is quantitatively the major tetrapyrrole in plants, with heme and other tetrapyrroles being present in minor amounts. Legume root nodules represent an exception, in which heme is synthesized in high quantity in the absence of chlorophyll, thus requiring the activity of enzymes not normally expressed highly in nonphotosynthetic tissues. Heme is synthesized from the universal tetrapyrrole precursor ALA by seven successive enzymatic steps; chlorophyll formation diverges after the synthesis of protoporphyrin, the immediate heme precursor (for review, see O''Brian, 1996). Biochemical and genetic evidence shows that soybean heme biosynthesis genes are strongly induced in root nodules (Sangwan and O''Brian, 1991, 1992, 1993; Madsen et al., 1993; Kaczor et al., 1994; Frustaci et al., 1995; Santana et al., 1998), and immunohistochemical studies demonstrate that induction is concentrated in infected nodule cells (Santana et al., 1998).ALA is synthesized from Glu in plants by a three-step mechanism called the C₅ pathway (Fig. ​(Fig.1);1); the latter two steps are committed to ALA synthesis and are catalyzed by GTR and GSAT, respectively (for review, see Beale and Weinstein, 1990; Jahn et al., 1991). Plant cDNA or genes encoding GTR (Gtr, also called HemA) and GSAT (Gsa) have been identified in several plant species (Grimm, 1990; Sangwan and O''Brian, 1993; Hofgen et al., 1994; Ilag et al., 1994; Frustaci et al., 1995; Wenzlau and Berry-Lowe, 1995; Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). Two genes for each enzyme have been described, and some genes are reported to be specific to a tissue, tetrapyrrole, or light regimen (Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). However, soybean Gsa1 is highly expressed in both leaves and nodules and contains a cis-acting element in its promoter that binds to a nuclear factor found in both tissues. (Frustaci et al., 1995). In this study we isolated soybean Gtr1 and characterized the genetic basis of GTR expression in root nodules. Figure 1C₅ pathway for ALA synthesis. The committed steps for ALA synthesis catalyzed by GTR and GSAT are boxed. Glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNA^Glu also participate in protein synthesis. The gene designations in plants are shown in parentheses ...     

Activity of the Fe-Branch of Tetrapyrrole Biosynthesis in the Chlorophyll-Deficient Plastome Mutant of Sunflower

The biosynthesis of heme, a plant tetrapyrrole, was studied in the leaves of a chlorophyll-deficient plastome mutant of the sunflower (Helianthus annuus L, line 2-24, albina form). In the light, the content of 5-aminolevulinic acid (ALA) in white mutant leaves was, on the average, ten times less than in that of the wild-type form (line 3629). Chlorophyll content in mutant leaves comprised only 0.3% of that of control plants. The activities of Fe-chelatase and ALA dehydratase in the heme synthesis were either comparable to or even higher than those in the wild-type leaves. A normal respiration rate in white mutant leaves, the equal content of phytochrome apoproteins in plants of both types, and the lack of noticeable morphogenetic differences realized through the phytochrome system can indicate that mutant and wild-type leaves are similar in their levels of phytochrome and the cytochromes of mitochondrial respiration. Nevertheless, in the mutant, the content of heme noncovalently bound by apoproteins amounted to only one third of its content in the wild-type plants. It seems that a dramatic decrease in the capability of white leaves for chlorophyll biosynthesis and for the formation of the photosynthetic apparatus is responsible for a low demand for chloroplast cytochromes, which is the major cause of a reduced heme content in the mutant.     

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.     

In vivo functional analysis of the structural domains of FLUORESCENT (FLU)

The control of chlorophyll (Chl) synthesis in angiosperms depends on the light-operating enzyme protochlorophyllide oxidoreductase (POR). The interruption of Chl synthesis during darkness requires suppression of the synthesis of 5-aminolevulinic acid (ALA), the first precursor molecule specific for Chl synthesis. The inactivation of glutamyl-tRNA reductase (GluTR), the first enzyme in tetrapyrrole biosynthesis, accomplished the decreased ALA synthesis by the membrane-bound protein FLUORESCENT (FLU) and prevents overaccumulation of protochlorophyllide (Pchlide) in the dark. We set out to elucidate the molecular mechanism of FLU-mediated inhibition of ALA synthesis, and explored the role of each of the three structural domains of mature FLU, the transmembrane, coiled-coil and tetratricopeptide repeat (TPR) domains, in this process. Efforts to rescue the FLU knock-out mutant with truncated FLU peptides revealed that, on its own, the TPR domain is insufficient to inactivate GluTR, although tight binding of the TPR domain to GluTR was detected. A truncated FLU peptide consisting of transmembrane and TPR domains also failed to inactivate GluTR in the dark. Similarly, suppression of ALA synthesis could not be achieved by combining the coiled-coil and TPR domains. Interaction studies revealed that binding of GluTR and POR to FLU is essential for inhibiting ALA synthesis. These results imply that all three FLU domains are required for the repression of ALA synthesis, in order to avoid the overaccumulation of Pchlide in the dark. Only complete FLU ensures the formation of a membrane-bound ternary complex consisting at least of FLU, GluTR and POR to repress ALA synthesis.     

Regulation of 5-aminolevulinic Acid synthesis in developing chloroplasts : I. Effect of light/dark treatments in vivo and in organello

Intact chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons regenerated protochlorophyllide (Pchlide) in the dark with added cofactors from either exogenous glutamate or endogenous substrates. No other intermediates of the chlorophyll biosynthetic pathway accumulated. When inhibitors of 5-aminolevulinic acid (ALA) dehydratase were added, the Pchlide that failed to form was replaced by an excessive amount of ALA. When greening seedlings were returned to the dark, ALA-synthesizing activity in the isolated chloroplasts decreased dramatically and recovered if the dark-treated seedlings were again exposed to continuous white light prior to chloroplast isolation. Both the decline and the recovery of ALA-synthesizing activity were complete in approximately 50 minutes. Changes in chloroplast structure during in vivo light to dark and dark to light transitions (as evidenced by electron microscopy) were much slower. Exposing isolated chloroplasts from dark-treated seedlings to short white flashes before incubation transformed nearly all the endogenous Pchlide, but hardly stimulated ALA synthesis, suggesting that Pchlide does not act as a feed-back inhibitor on ALA synthesis. Chloroplasts isolated from dark-treated tissue did not form Pchlide from glutamate when incubated in the dark with added cofactors; moreover, the endogenous Pchlide did not turn over in organello. However, these chloroplasts did synthesize Pchlide from added ALA at the normal rate and synthesized ALA from glutamate at a reduced, but still significant, rate. Mg chelation was not affected by in vivo dark treatment.     

The mapping of phytochrome genes and photomorphogenic mutants of tomato

The map positions of five previously described phytochrome genes have been determined in tomato (Lycopersicon esculentum Mill.) The position of the yg-2 gene on chromosome 12 has been confirmed and the classical map revised. The position of the phytochrome A (phy A)-deficient fri mutants has been refined by revising the classical map of chromosome 10. The position of the PhyA gene is indistinguishable from that of the fri locus. The putative phyB1-deficient tri mutants were mapped by classical and RFLP analysis to chromosome 1. The PhyB1 gene, as predicted, was located at the same position. Several mutants with the high pigment (hp) phenotype, which exaggerates phytochrome responses, have been reported. Allelism tests confirmed that the hp-2 mutant is not allelic to other previously described hp (proposed here to be called hp-1) mutants and a second stronger hp-2 allele (hp-2 ^j ) was identified. The hp-2 gene was mapped to the classical, as well as the RFLP, map of chromosome 1. Received: 24 May 1996 / Accepted: 14 June 1996     

Recent advances in microbial production of δ-aminolevulinic acid and vitamin B12

δ-aminolevulinate (ALA) is an important intermediate involved in tetrapyrrole synthesis (precursor for vitamin B₁₂, chlorophyll and heme) in vivo. It has been widely applied in agriculture and medicine. On account of many disadvantages of its chemical synthesis, microbial production of ALA has been received much attention as an alternative because of less expensive raw materials, low pollution, and high productivity. Vitamin B₁₂, one of ALA derivatives, which plays a vital role in prevention of anaemia has also attracted intensive works. In this review, recent advances on the production of ALA and vitamin B₁₂ with novel approaches such as whole-cell enzyme-transformation and metabolic engineering are described. Furthermore, the direction for future research and perspective are also summarized.