A Genetically Encoded Fluorescent Heme Sensor Detects Free Heme in Plants.

Heme is produced in plants via a plastid-localized metabolic pathway and is subsequently distributed to all cellular compartments. In addition to covalently and non-covalently bound heme, a comparatively small amount of free heme that is not associated with protein is available for incorporation into heme-dependent proteins in all subcellular compartments and for regulatory purposes. This "labile" fraction may also be toxic. To date, the distribution of the free heme pool in plant cells remains poorly understood. Several fluorescence-based methods for the quantification of intracellular free heme have been described. For this study, we used the previously described genetically encoded heme sensor 1 (HS1) to measure the relative amounts of heme in different plant subcellular compartments. In a proof of concept, we manipulated heme content using a range of biochemical and genetic approaches and verified the utility of HS1 in different cellular compartments of Arabidopsis (Arabidopsis thaliana) and tobacco (N. tabacum and N. benthamiana) plants transformed either transiently or stably with HS1 and HS1(M7A), a variant with lower affinity for heme. This approach makes it possible to trace the distribution and dynamics of free heme and provides relevant information about its mobilization. The application of these heme sensors will create opportunities to explore and validate the importance of free heme in plant cells and to identify mutants that alter the subcellular allocation of free heme.

More indications for redox-sensitive cysteine residues of the Arabidopsis 5-aminolevulinate dehydratase.

Redox-dependent thiol-disulfide switches of cysteine residues are one of the significant posttranslational modifications of proteins to control rapidly their stability, activity, and protein interaction. Redox control also modulates the tetrapyrrole biosynthesis (TBS). Among the redox-dependent TBS enzymes, 5-aminolevulinic acid dehydratase (ALAD) was previously recognized to interact with reductants, such a thioredoxins or NADPH-dependent thioredoxin reductase C. In this report, we aim to verify the redox sensitivity of ALAD and identify the redox-reactive cysteine residues among the six cysteines of the mature protein form Arabidopsis. Based on structural modelling and comparative studies of wild-type ALAD and ALAD mutants with single and double Cys➔Ser substitutions under oxidizing and reducing conditions, we aim to predict the dimerization and oligomerisation of ALAD as well as the crucial Cys residues for disulfide bridge formation and enzyme activity. The Cys404Ser mutation led to a drastic inactivation of ALAD and redox-dependent properties of ALAD were severely impaired, when Cys71 was simultaneously mutated with Cys152 or Cys251. Cys71 is located in a flexible N-terminal arm of ALAD, which could allow intramolecular disulfide bridges with Cys residues at the surface of the remaining globule ALAD structure. As a result, we propose different roles of Cys residues for redox control, catalytic activity and Mg2+-dependent assembly.

The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron-sulfur cluster biogenesis.

Fe-S clusters are ancient, ubiquitous and highly essential prosthetic groups for numerous fundamental processes of life. The biogenesis of Fe-S clusters is a multistep process including iron acquisition, sulfur mobilization, and cluster formation. Extensive studies have provided deep insights into the mechanism of the latter two assembly steps. However, the mechanism of iron utilization during chloroplast Fe-S cluster biogenesis is still unknown. Here we identified two Arabidopsis DnaJ proteins, DJA6 and DJA5, that can bind iron through their conserved cysteine residues and facilitate iron incorporation into Fe-S clusters by interactions with the SUF (sulfur utilization factor) apparatus through their J domain. Loss of these two proteins causes severe defects in the accumulation of chloroplast Fe-S proteins, a dysfunction of photosynthesis, and a significant intracellular iron overload. Evolutionary analyses revealed that DJA6 and DJA5 are highly conserved in photosynthetic organisms ranging from cyanobacteria to higher plants and share a strong evolutionary relationship with SUFE1, SUFC, and SUFD throughout the green lineage. Thus, our work uncovers a conserved mechanism of iron utilization for chloroplast Fe-S cluster biogenesis.

Acclimation in plants - the Green Hub consortium.

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to 'smart breeding' methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast-related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.

Post-translational coordination of chlorophyll biosynthesis and breakdown by BCMs maintains chlorophyll homeostasis during leaf development.

Chlorophyll is indispensable for life on Earth. Dynamic control of chlorophyll level, determined by the relative rates of chlorophyll anabolism and catabolism, ensures optimal photosynthesis and plant fitness. How plants post-translationally coordinate these two antagonistic pathways during their lifespan remains enigmatic. Here, we show that two Arabidopsis paralogs of BALANCE of CHLOROPHYLL METABOLISM (BCM) act as functionally conserved scaffold proteins to regulate the trade-off between chlorophyll synthesis and breakdown. During early leaf development, BCM1 interacts with GENOMES UNCOUPLED 4 to stimulate Mg-chelatase activity, thus optimizing chlorophyll synthesis. Meanwhile, BCM1's interaction with Mg-dechelatase promotes degradation of the latter, thereby preventing chlorophyll degradation. At the onset of leaf senescence, BCM2 is up-regulated relative to BCM1, and plays a conserved role in attenuating chlorophyll degradation. These results support a model in which post-translational regulators promote chlorophyll homeostasis by adjusting the balance between chlorophyll biosynthesis and breakdown during leaf development.

Characterization of a Plastoglobule-Localized SOUL4 Heme-Binding Protein in Arabidopsis thaliana.

Heme plays an active role in primary plant metabolic pathways as well as in stress signaling. In this study, we characterized the predicted heme-binding protein SOUL4. Proteomics evidence suggests that SOUL4 is a component of Arabidopsis plastoglobules (PGs, chloroplast lipid droplets). SOUL4 contains heme-binding motifs and the recombinant protein is shown here to bind heme in vitro. Fluorescence-tagged SOUL4 colocalized with the specific PG marker Fibrillin1A (FBN1A) in transiently transformed Nicotiana benthamiana leaves. In addition, SOUL4 cofractionated with another PG marker Fibrillin2 (FBN2) in sucrose gradient ultracentrifugation experiments. In vitro kinase experiments revealed that SOUL4 is phosphorylated by a yet unknown chloroplast protein kinase. Our data demonstrate that SOUL4 is a bona fide PG protein and may function in heme-buffering in the chloroplast.

Redox-control of chlorophyll biosynthesis mainly depends on thioredoxins.

In order to maintain enzyme stability and activity, chloroplasts use two systems of thiol-disulfide reductases for the control of redox-dependent properties of proteins. Previous studies have revealed that plastid-localized thioredoxins (TRX) and the NADPH-dependent thioredoxin reductase C (NTRC) are important for the reduction of cysteine residues of enzymes involved in chlorophyll synthesis. Very recently, it was shown that the pale green phenotype of the ntrc mutant is suppressed when the contents of 2-cysteine peroxiredoxins (2CP) A and B are decreased. Here, we show that suppression of the ntrc phenotype results from a recovery of wild-type-like redox control of chlorophyll biosynthesis enzymes in ntrc/2cp mutants. The presented results support the conclusion that TRXs rather than NTRC are the predominant reductases mediating the redox-regulation of these enzymes.

Transgenic Tobacco Lines Expressing Sense or Antisense FERROCHELATASE 1 RNA Show Modified Ferrochelatase Activity in Roots and Provide Experimental Evidence for Dual Localization of Ferrochelatase 1.

In plants, two genes encode ferrochelatase (FC), which catalyzes iron chelation into protoporphyrin IX at the final step of heme biosynthesis. FERROCHELATASE1 (FC1) is continuously, but weakly expressed in roots and leaves, while FC2 is dominantly active in leaves. As a continuation of previous studies on the physiological consequences of FC2 inactivation in tobacco, we aimed to assign FC1 function in plant organs. While reduced FC2 expression leads to protoporphyrin IX accumulation in leaves, FC1 down-regulation and overproduction caused reduced and elevated FC activity in root tissue, respectively, but were not associated with changes in macroscopic phenotype, plant development or leaf pigmentation. In contrast to the lower heme content resulting from a deficiency of the dominant FC2 expression in leaves, a reduction of FC1 in roots and leaves does not significantly disturb heme accumulation. The FC1 overexpression was used for an additional approach to re-examine FC activity in mitochondria. Transgenic FC1 protein was immunologically shown to be present in mitochondria. Although matching only a small portion of total cellular FC activity, the mitochondrial FC activity in a FC1 overexpressor line increased 5-fold in comparison with wild-type mitochondria. Thus, it is suggested that FC1 contributes to mitochondrial heme synthesis.

Identification of four plastid-localized protein kinases.

In chloroplasts, protein phosphorylation regulates important processes, including metabolism, photosynthesis, gene expression, and signaling. Because the hitherto known plastid protein kinases represent only a fraction of existing kinases, we aimed at the identification of novel plastid-localized protein kinases that potentially phosphorylate enzymes of the tetrapyrrole biosynthesis (TBS) pathway. We screened publicly available databases for proteins annotated as putative protein kinase family proteins with predicted chloroplast localization. Additionally, we analyzed chloroplast fractions which were separated by sucrose density gradient centrifugation by mass spectrometry. We identified four new candidates for protein kinases, which were confirmed to be plastid localized by expression of GFP-fusion proteins in tobacco leaves. A phosphorylation assay with the purified kinases confirmed the protein kinase activity for two of them.

Arabidopsis Mg-Protoporphyrin IX Methyltransferase Activity and Redox Regulation Depend on Conserved Cysteines.

Redox regulation is an essential post-translational regulatory mechanism in prokaryotes and eukaryotes. The reversible oxidation and reduction of cysteine residues of proteins is also important in photosynthetic organisms to control enzymatic activities, protein stability and the interaction with other proteins of chloroplast-localized proteins. Several enzymes of the plant tetrapyrrole biosynthesis pathway have been identified to be redox regulated by thioredoxins (TRXs) and NADPH-dependent thioredoxin reductase C (NTRC). Among these proteins, Mg protoporphyrin IX methyltransferase (encoded by CHLM) was identified to be activated and stabilized by interaction with NTRC. CHLM catalyzes a methyl group transfer by using S-adenosylmethionine (SAM). Here we demonstrate that three conserved cysteine residues of Arabidopsis CHLM are essential for catalytic function and redox-dependent activation of the enzyme. In vitro and in planta biochemical assays of recombinant CHLM and the Arabidopsis chlm knockout mutant overexpressing wild-type and cysteine substitution mutants of CHLM revealed modified methyltransferase activity, when the conserved cysteine residues of CHLM are replaced by serine. While C177 is responsible for redox-dependent enzyme activation, exchange of the two cysteine residues, C111 and C115, has a strong impact on enzyme activity. The modified CHLM activity of single and double mutants with cysteine substitution is presented, and the role of each cysteine residue is discussed based on a modeled structure of CHLM. These studies contribute to enhanced understanding of the physiological and enzymatic significance of redox-regulated CHLM.

Pea aphid infestation induces changes in flavonoids, antioxidative defence, soluble sugars and sugar transporter expression in leaves of pea seedlings.

The perception of aphid infestation induces highly coordinated and sequential defensive reactions in plants at the cellular and molecular levels. The aim of the study was to explore kinetics of induced antioxidative defence responses in leaf cells of Pisum sativum L.cv. Cysterski upon infestation of the pea aphid Acyrthosiphon pisum at varying population sizes, including accumulation of flavonoids, changes of carbon metabolism, and expression of nuclear genes involved in sugar transport. Within the first 96 h, after A. pisum infestation, flavonoid accumulation and increased peroxidase activity were observed in leaves. The level of pisatin increased after 48 h of infestation and reached a maximum at 96 h. At this time point, a higher concentration of flavonols was observed in the infested tissue than in the control. Additionally, strong post-infestation accumulation of chalcone synthase (CHS) and isoflavone synthase (IFS) transcription products was also found. The levels of sucrose and fructose in 24-h leaves infested by 10, 20, and 30 aphids were significantly lower than in the control. Moreover, in leaves infested by 30 aphids, the reduced sucrose level observed up to 48 h was accompanied by a considerable increase in the expression level of the PsSUT1 gene encoding the sucrose transporter. In conclusion, A. pisum infestation on pea leads to stimulation of metabolic pathways associated with defence.

Production of ketocarotenoids in tobacco alters the photosynthetic efficiency by reducing photosystem II supercomplex and LHCII trimer stability.

The consequences of ketocarotenoid production in transgenic tobacco (Nicotiana tabacum) plants expressing a Chlamydomonas reinhardtii gene encoding a ß-carotene ketolase were examined concerning the functionality of the photosynthetic apparatus. T1 plants produced less photosynthetic pigments per dry weight, but Chl a/Chl b ratios remained unchanged. Almost as much ketocarotenoids as accessory xanthophylls accumulated per Chl a molecule. These ketocarotenoids were found mainly in the thylakoid membranes, but were not functionally bound to light-harvesting complexes, although LHCII is known to be able to bind astaxanthin. On the contrary, high amounts of ketocarotenoids probably changed the properties of the lipid phase of the thylakoids, thereby reducing the stability of photosystem II supercomplexes and LHCII trimers and ultimately decreasing grana formation. In addition, photosystem II function in electron transport was impaired, and plants exhibited less non-photochemical quenching compared to wild-type plants. Thus, in order not to disturb vital functions of the plants, production of astaxanthin and other nutritionally valuable ketocarotenoids apparently requires ways to sequester the additional carotenoids to plastoglobuli.

Site directed mutagenesis of StSUT1 reveals target amino acids of regulation and stability.

Plant sucrose transporters (SUTs) are functional as sucrose-proton-cotransporters with an optimal transport activity in the acidic pH range. Recently, the pH optimum of the Solanum tuberosum sucrose transporter StSUT1 was experimentally determined to range at an unexpectedly low pH of 3 or even below. Various research groups have confirmed these surprising findings independently and in different organisms. Here we provide further experimental evidence for a pH optimum at physiological extrema. Site directed mutagenesis provides information about functional amino acids, which are highly conserved and responsible for this extraordinary increase in transport capacity under extreme pH conditions. Redox-dependent dimerization of the StSUT1 protein was described earlier. Here the ability of StSUT1 to form homodimers was demonstrated by heterologous expression in Lactococcus lactis and Xenopus leavis using Western blots, and in plants by bimolecular fluorescence complementation. Mutagenesis of highly conserved cysteine residues revealed their importance in protein stability. The accessibility of regulatory amino acid residues in the light of StSUT1's compartmentalization in membrane microdomains is discussed.

Posttranslational influence of NADPH-dependent thioredoxin reductase C on enzymes in tetrapyrrole synthesis.

The NADPH-dependent thioredoxin reductase C (NTRC) is involved in redox-related regulatory processes in chloroplasts and nonphotosynthetic active plastids. Together with 2-cysteine peroxiredoxin, it forms a two-component peroxide-detoxifying system that acts as a reductant under stress conditions. NTRC stimulates in vitro activity of magnesium protoporphyrin IX monomethylester (MgPMME) cyclase, most likely by scavenging peroxides. Reexamination of tetrapyrrole intermediate levels of the Arabidopsis (Arabidopsis thaliana) knockout ntrc reveals lower magnesium protoporphyrin IX (MgP) and MgPMME steady-state levels, the substrate and the product of MgP methyltransferase (CHLM) preceding MgPMME cyclase, while MgP strongly accumulates in mutant leaves after 5-aminolevulinic acid feeding. The ntrc mutant has a reduced capacity to synthesize 5-aminolevulinic acid and reduced CHLM activity compared with the wild type. Although transcript levels of genes involved in chlorophyll biosynthesis are not significantly altered in 2-week-old ntrc seedlings, the contents of glutamyl-transfer RNA reductase1 (GluTR1) and CHLM are reduced. Bimolecular fluorescence complementation assay confirms a physical interaction of NTRC with GluTR1 and CHLM. While ntrc contains partly oxidized CHLM, the wild type has only reduced CHLM. As NTRC also stimulates CHLM activity in vitro, it is proposed that NTRC has a regulatory impact on the redox status of conserved cysteine residues of CHLM. It is hypothesized that a deficiency of NTRC leads to a lower capacity to reduce cysteine residues of GluTR1 and CHLM, affecting the stability and, thereby, altering the activity in the entire tetrapyrrole synthesis pathway.

Photoperiodic regulation of the sucrose transporter StSUT4 affects the expression of circadian-regulated genes and ethylene production.

Several recent publications reported different subcellular localization of the sucrose transporters belonging to the SUT4 subfamily. The physiological function of the SUT4 sucrose transporters requires clarification, because down-regulation of the members of the SUT4 clade had different effects in rice, poplar, and potato. Here, we provide new data for the localization and function of the Solanaceous StSUT4 protein, further elucidating involvement in the onset of flowering, tuberization and in the shade avoidance syndrome of potato plants. Induction of an early flowering and a tuberization in the SUT4-inhibited potato plants correlates with increased sucrose export from leaves and increased sucrose and starch accumulation in terminal sink organs, such as developing tubers. SUT4 affects expression of the enzymes involved in gibberellin and ethylene biosynthesis, as well as the rate of ethylene biosynthesis in potato. In the SUT4-inhibited plants, the ethylene production no longer follows a diurnal rhythm. Thus it was concluded that StSUT4 controls circadian gene expression, potentially by regulating sucrose export from leaves. Furthermore, SUT4 expression affects clock-regulated genes such as StFT, StSOC1, and StCO, which might be also involved in a photoperiod-dependent tuberization. A model is proposed in which StSUT4 controls a phloem-mobile signaling molecule generated in leaves, which together with enhanced sucrose export affects developmental switches in apical meristems. SUT4 seems to link photoreceptor-perceived information about the light quality and day length with phytohormone biosynthesis and the expression of circadian-regulated genes.

LCAA, a novel factor required for magnesium protoporphyrin monomethylester cyclase accumulation and feedback control of aminolevulinic acid biosynthesis in tobacco.

Low Chlorophyll Accumulation A (LCAA) antisense plants were obtained from a screen for genes whose partial down-regulation results in a strong chlorophyll deficiency in tobacco (Nicotiana tabacum). The LCAA mutants are affected in a plastid-localized protein of unknown function, which is conserved in cyanobacteria and all photosynthetic eukaryotes. They suffer from drastically reduced light-harvesting complex (LHC) contents, while the accumulation of all other photosynthetic complexes per leaf area is less affected. As the disturbed accumulation of LHC proteins could be either attributable to a defect in LHC biogenesis itself or to a bottleneck in chlorophyll biosynthesis, chlorophyll synthesis rates and chlorophyll synthesis intermediates were measured. LCAA antisense plants accumulate magnesium (Mg) protoporphyrin monomethylester and contain reduced protochlorophyllide levels and a reduced content of CHL27, a subunit of the Mg protoporphyrin monomethylester cyclase. Bimolecular fluorescence complementation assays confirm a direct interaction between LCAA and CHL27. 5-Aminolevulinic acid synthesis rates are increased and correlate with an increased content of glutamyl-transfer RNA reductase. We suggest that LCAA encodes an additional subunit of the Mg protoporphyrin monomethylester cyclase, is required for the stability of CHL27, and contributes to feedback-control of 5-aminolevulinic acid biosynthesis, the rate-limiting step of chlorophyll biosynthesis.

Homologous NF-YC2 subunit from Arabidopsis and tobacco is activated by photooxidative stress and induces flowering.

The transcription factor NF-Y consists of the three subunits A, B and C, which are encoded in Arabidopsis in large gene families. The multiplicity of the genes implies that NF-Y may act in diverse combinations of each subunit for the transcriptional control. We aimed to assign a function in stress response and plant development to NF-YC subunits by analyzing the expression of NF-Y genes and exploitation of nf-y mutants. Among the subunit family, NF-YC2 showed the strongest inducibility towards oxidative stress, e.g. photodynamic, light, oxidative, heat and drought stress. A tobacco NF-YC homologous gene was found to be inducible by photooxidative stress generated by an accumulation of the tetrapyrrole metabolite, coproporphyrin. Despite the stress induction, an Arabidopsis nf-yc2 mutant and NF-YC2 overexpressors did not show phenotypical differences compared to wild-type seedlings in response to photooxidative stress. This can be explained by the compensatory potential of other members of the NF-YC family. However, NF-YC2 overexpression leads to an early flowering phenotype that is correlated with increased FLOWERING LOCUS T-transcript levels. It is proposed that NF-YC2 functions in floral induction and is a candidate gene among the NF-Y family for the transcriptional activation upon oxidative stress.

Thioredoxin redox regulates ATPase activity of magnesium chelatase CHLI subunit and modulates redox-mediated signaling in tetrapyrrole biosynthesis and homeostasis of reactive oxygen species in pea plants.

The chloroplast thioredoxins (TRXs) function as messengers of redox signals from ferredoxin to target enzymes. In this work, we studied the regulatory impact of pea (Pisum sativum) TRX-F on the magnesium (Mg) chelatase CHLI subunit and the enzymatic activation of Mg chelatase in vitro and in vivo. In vitro, reduced TRX-F activated the ATPase activity of pea CHLI and enhanced the activity of Mg chelatase reconstituted from the three recombinant subunits CHLI, CHLD, and CHLH in combination with the regulator protein GENOMES UNCOUPLED4 (GUN4). Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that TRX-F physically interacts with CHLI but not with either of the other two subunits or GUN4. In vivo, virus-induced TRX-F gene silencing (VIGS-TRX-F) in pea plants did not result in an altered redox state of CHLI. However, simultaneous silencing of the pea TRX-F and TRX-M genes (VIGS-TRX-F/TRX-M) resulted in partially and fully oxidized CHLI in vivo. VIGS-TRX-F/TRX-M plants demonstrated a significant reduction in Mg chelatase activity and 5-aminolevulinic acid synthesizing capacity as well as reduced pigment content and lower photosynthetic capacity. These results suggest that, in vivo, TRX-M can compensate for a lack of TRX-F and that both TRXs act as important redox regulators of Mg chelatase. Furthermore, the silencing of TRX-F and TRX-M expression also affects gene expression in the tetrapyrrole biosynthesis pathway and leads to the accumulation of reactive oxygen species, which may also serve as an additional signal for the transcriptional regulation of photosynthesis-associated nuclear genes.

Studies on differential nuclear translocation mechanism and assembly of the three subunits of the Arabidopsis thaliana transcription factor NF-Y.

The eukaryotic transcription factor NF-Y consists of three subunits (A, B, and C), which are encoded in Arabidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combinations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF-YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.

An Arabidopsis GluTR binding protein mediates spatial separation of 5-aminolevulinic acid synthesis in chloroplasts.

5-Aminolevulinic acid (ALA) is the universal precursor for tetrapyrrole biosynthesis and is synthesized in plants in three enzymatic steps: ligation of glutamate (Glu) to tRNA(Glu) by glutamyl-tRNA synthetase, reduction of activated Glu to Glu-1-semialdehyde by glutamyl-tRNA reductase (GluTR), and transamination to ALA by Glu 1-semialdehyde aminotransferase. ALA formation controls the metabolic flow into the tetrapyrrole biosynthetic pathway. GluTR is proposed to be the key regulatory enzyme that is tightly controlled at transcriptional and posttranslational levels. We identified a GluTR binding protein (GluTRBP; previously called PROTON GRADIENT REGULATION7) that is localized in chloroplasts and part of a 300-kD protein complex in the thylakoid membrane. Although the protein does not modulate activity of ALA synthesis, the knockout of GluTRBP is lethal in Arabidopsis thaliana, whereas mutants expressing reduced levels of GluTRBP contain less heme. GluTRBP expression correlates with a function in heme biosynthesis. It is postulated that GluTRBP contributes to subcompartmentalized ALA biosynthesis by maintaining a portion of GluTR at the plastid membrane that funnels ALA into the heme biosynthetic pathway. These results regarding GluTRBP support a model of plant ALA synthesis that is organized in two separate ALA pools in the chloroplast to provide appropriate substrate amounts for balanced synthesis of heme and chlorophyll.