Directing cyanobacterial photosynthesis in a cytochrome c oxidase mutant using a heterologous electron sink.

Photosynthesis holds the promise of sustainable generation of useful products using light energy. Key to realizing this potential is the ability to rationally design photosynthesis to redirect energy and reductant derived from photons to desired products. Cytochrome P450s (P450s), which catalyze a broad array of reactions, have been engineered into a variety of photosynthetic organisms, where their activity has been shown to be photosynthesis-dependent, thus acting as heterologous sinks of electrons derived from photosynthesis. Furthermore, the addition of P450s can increase the photosynthetic capacity of the host organism. In this study, we developed this technology further using a P450 (CYP1A1) expressed in the cyanobacterium Synechococcus sp. PCC 7002. We show that rationally engineering photosynthesis by the removal of a competing electron sink, the respiratory terminal oxidase cytochrome c oxidase, increased the activity of CYP1A1. We provide evidence that this enhanced CYP1A1 activity was facilitated via an increase in the flux of electrons through Photosystem I. We also conducted a transcriptomic analysis on the designed strains to gain a more holistic understanding of how the cell responds to rational engineering. We describe a complex response including changes in expression of genes involved in photosynthesis and electron transfer linked to respiration. Specifically, the expression of CYP1A1 resulted in the reduction in expression of other natural electron dissipation pathways. This study emphasizes the potential for engineering photosynthetic organisms in biotechnology but also highlights the need to consider the broader impacts on cellular metabolism of any rationally induced changes.

The retrograde signaling protein GUN1 regulates tetrapyrrole biosynthesis.

The biogenesis of the photosynthetic apparatus in developing seedlings requires the assembly of proteins encoded on both nuclear and chloroplast genomes. To coordinate this process there needs to be communication between these organelles, but the retrograde signals by which the chloroplast communicates with the nucleus at this time are still essentially unknown. The Arabidopsis thaliana genomes uncoupled (gun) mutants, that show elevated nuclear gene expression after chloroplast damage, have formed the basis of our understanding of retrograde signaling. Of the 6 reported gun mutations, 5 are in tetrapyrrole biosynthesis proteins and this has led to the development of a model for chloroplast-to-nucleus retrograde signaling in which ferrochelatase 1 (FC1)-dependent heme synthesis generates a positive signal promoting expression of photosynthesis-related genes. However, the molecular consequences of the strongest of the gun mutants, gun1, are poorly understood, preventing the development of a unifying hypothesis for chloroplast-to-nucleus signaling. Here, we show that GUN1 directly binds to heme and other porphyrins, reduces flux through the tetrapyrrole biosynthesis pathway to limit heme and protochlorophyllide synthesis, and can increase the chelatase activity of FC1. These results raise the possibility that the signaling role of GUN1 may be manifested through changes in tetrapyrrole metabolism, supporting a role for tetrapyrroles as mediators of a single biogenic chloroplast-to-nucleus retrograde signaling pathway.

The iron-sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis, but not phytochrome signaling.

Proteins that contain iron-sulfur (Fe-S) clusters play pivotal roles in various metabolic processes such as photosynthesis and redox metabolism. Among the proteins involved in the biosynthesis of Fe-S clusters in plants, the SUFB subunit of the SUFBCD complex appears to be unique because SUFB has been reported to be involved in chlorophyll metabolism and phytochrome-mediated signaling. To gain insights into the function of the SUFB protein, we analyzed the phenotypes of two SUFB mutants, laf6 and hmc1, and RNA interference (RNAi) lines with reduced SUFB expression. When grown in the light, the laf6 and hmc1 mutants and the SUFB RNAi lines accumulated higher levels of the chlorophyll biosynthesis intermediate Mg-protoporphyrin IX monomethylester (Mg-proto MME), consistent with the impairment of Mg-proto MME cyclase activity. Both SUFC- and SUFD-deficient RNAi lines accumulated the same intermediate, suggesting that inhibition of Fe-S cluster synthesis is the primary cause of this impairment. Dark-grown laf6 seedlings also showed an increase in protoporphyrin IX (Proto IX), Mg-proto, Mg-proto MME and 3,8-divinyl protochlorophyllide a (DV-Pchlide) levels, but this was not observed in hmc1 or the SUFB RNAi lines, nor was it complemented by SUFB overexpression. In addition, the long hypocotyl in far-red light phenotype of the laf6 mutant could not be rescued by SUFB overexpression and segregated from the pale-green SUFB-deficient phenotype, indicating it is not caused by mutation at the SUFB locus. These results demonstrate that biosynthesis of Fe-S clusters is important for chlorophyll biosynthesis, but that the laf6 phenotype is not due to a SUFB mutation.

Singlet oxygen initiates a plastid signal controlling photosynthetic gene expression.

Retrograde signals from the plastid regulate photosynthesis-associated nuclear genes and are essential to successful chloroplast biogenesis. One model is that a positive haem-related signal promotes photosynthetic gene expression in a pathway that is abolished by the herbicide norflurazon. Far-red light (FR) pretreatment and transfer to white light also results in plastid damage and loss of photosynthetic gene expression. Here, we investigated whether norflurazon and FR pretreatment affect the same retrograde signal. We used transcriptome analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) to analyse the effects of these treatments on nuclear gene expression in various Arabidopsis (Arabidopsis thaliana) retrograde signalling mutants. Results showed that the two treatments inhibited largely different nuclear gene sets, suggesting that they affected different retrograde signals. Moreover, FR pretreatment resulted in singlet oxygen (1 O2 ) production and a rapid inhibition of photosynthetic gene expression. This inhibition was partially blocked in the executer1executer2 mutant, which is impaired in 1 O2 signalling. Our data support a new model in which a 1 O2 retrograde signal, generated by chlorophyll precursors, inhibits expression of key photosynthetic and chlorophyll synthesis genes to prevent photo-oxidative damage during de-etiolation. Such a signal would provide a counterbalance to the positive haem-related signal to fine tune regulation of chloroplast biogenesis.

A chloroplast-localized protein LESION AND LAMINA BENDING affects defence and growth responses in rice.

Understanding how plants allocate their resources to growth or defence is of long-term importance to the development of new and improved varieties of different crops. Using molecular genetics, plant physiology, hormone analysis and Next-Generation Sequencing (NGS)-based transcript profiling, we have isolated and characterized the rice (Oryza sativa) LESION AND LAMINA BENDING (LLB) gene that encodes a chloroplast-targeted putative leucine carboxyl methyltransferase. Loss of LLB function results in reduced growth and yield, hypersensitive response (HR)-like lesions, accumulation of the antimicrobial compounds momilactones and phytocassanes, and constitutive expression of pathogenesis-related genes. Consistent with these defence-associated responses, llb shows enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae). The lesion and resistance phenotypes are likely to be caused by the over-accumulation of jasmonates (JAs) in the llb mutant including the JA precursor 12-oxo-phytodienoic acid. Additionally, llb shows an increased lamina inclination and enhanced early seedling growth due to elevated brassinosteroid (BR) synthesis and/or signalling. These findings show that LLB functions in the chloroplast to either directly or indirectly repress both JA- and BR-mediated responses, revealing a possible mechanism for controlling how plants allocate resources for defence and growth.

G protein-coupled receptor-type G proteins are required for light-dependent seedling growth and fertility in Arabidopsis.

G protein-coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein-coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1(pro):GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development.

The tillering phenotype of the rice plastid terminal oxidase (PTOX) loss-of-function mutant is associated with strigolactone deficiency.

The significance of plastid terminal oxidase (PTOX) in phytoene desaturation and chloroplast function has been demonstrated using PTOX-deficient mutants, particularly in Arabidopsis. However, studies on its role in monocots are lacking. Here, we report cloning and characterization of the rice (Oryza sativa) PTOX1 gene. Using Ecotype Targeting Induced Local Lesions IN Genomes (EcoTILLING) and TILLING as forward genetic tools, we identified the causative mutation of an EMS mutant characterized by excessive tillering, semi-dwarfism and leaf variegation that corresponded to the PTOX1 gene. The tillering and semi-dwarf phenotypes of the ptox1 mutant are similar to phenotypes of known strigolactone (SL)-related rice mutants, and both phenotypic traits could be rescued by application of the synthetic SL GR24. The ptox1 mutant accumulated phytoene in white leaf sectors with a corresponding deficiency in ß-carotene, consistent with the expected function of PTOX1 in promoting phytoene desaturase activity. There was also no accumulation of the carotenoid-derived SL ent-2'-epi-5-deoxystrigol in root exudates. Elevated concentrations of auxin were detected in the mutant, supporting previous observations that SL interaction with auxin is important in shoot branching control. Our results demonstrate that PTOX1 is required for both carotenoid and SL synthesis resulting in SL-deficient phenotypes in rice.

DELLAs regulate chlorophyll and carotenoid biosynthesis to prevent photooxidative damage during seedling deetiolation in Arabidopsis.

In plants, light represents an important environmental signal that triggers the production of photosynthetically active chloroplasts. This developmental switch is critical for plant survival because chlorophyll precursors that accumulate in darkness can be extremely destructive when illuminated. Thus, plants have evolved mechanisms to adaptively control plastid development during the transition into light. Here, we report that the gibberellin (GA)-regulated DELLA proteins play a crucial role in the formation of functional chloroplasts during deetiolation. We show that Arabidopsis thaliana DELLAs accumulating in etiolated cotyledons derepress chlorophyll and carotenoid biosynthetic pathways in the dark by repressing the transcriptional activity of the phytochrome-interacting factor proteins. Accordingly, dark-grown GA-deficient ga1-3 mutants (that accumulate DELLAs) display a similar gene expression pattern to wild-type seedlings grown in the light. Consistent with this, ga1-3 seedlings accumulate higher amounts of protochlorophyllide (a phototoxic chlorophyll precursor) in darkness but, surprisingly, are substantially more resistant to photooxidative damage following transfer into light. This is due to the DELLA-dependent upregulation of the photoprotective enzyme protochlorophyllide oxidoreductase (POR) in the dark. Our results emphasize the role of DELLAs in regulating the levels of POR, protochlorophyllide, and carotenoids in the dark and in protecting etiolated seedlings against photooxidative damage during initial light exposure.

PIF3 is a repressor of chloroplast development.

The phytochrome-interacting factor PIF3 has been proposed to act as a positive regulator of chloroplast development. Here, we show that the pif3 mutant has a phenotype that is similar to the pif1 mutant, lacking the repressor of chloroplast development PIF1, and that a pif1pif3 double mutant has an additive phenotype in all respects. The pif mutants showed elevated protochlorophyllide levels in the dark, and etioplasts of pif mutants contained smaller prolamellar bodies and more prothylakoid membranes than corresponding wild-type seedlings, similar to previous reports of constitutive photomorphogenic mutants. Consistent with this observation, pif1, pif3, and pif1pif3 showed reduced hypocotyl elongation and increased cotyledon opening in the dark. Transfer of 4-d-old dark-grown seedlings to white light resulted in more chlorophyll synthesis in pif mutants over the first 2 h, and analysis of gene expression in dark-grown pif mutants indicated that key tetrapyrrole regulatory genes such as HEMA1 encoding the rate-limiting step in tetrapyrrole synthesis were already elevated 2 d after germination. Circadian regulation of HEMA1 in the dark also showed reduced amplitude and a shorter, variable period in the pif mutants, whereas expression of the core clock components TOC1, CCA1, and LHY was largely unaffected. Expression of both PIF1 and PIF3 was circadian regulated in dark-grown seedlings. PIF1 and PIF3 are proposed to be negative regulators that function to integrate light and circadian control in the regulation of chloroplast development.

Tetrapyrrole profiling in Arabidopsis seedlings reveals that retrograde plastid nuclear signaling is not due to Mg-protoporphyrin IX accumulation.

Chloroplast biogenesis involves careful coordination of both plastid and nuclear gene expression, which is achieved in part by retrograde signaling from the chloroplast to the nucleus. This can be demonstrated by the fact that the herbicide, Norflurazon (NF), which causes bleaching of chloroplasts, prevents the light induction of photosynthesis-related genes in the nucleus. It has been proposed that the tetrapyrrole pathway intermediate Mg-protoporphyrin IX acts as the signaling molecule in this pathway and accumulates in the chloroplasts and cytosol of the cell after NF treatment. Here we present data that demonstrate that this model is too simplistic. We have developed a sensitive liquid chromatography-mass spectrometry (LC/MS) method to measure tetrapyrrole intermediates and have shown that no Mg-protoporphyrin IX, nor indeed any other chlorophyll-biosynthesis intermediate, can be detected in NF-treated plants under conditions in which nuclear gene expression is repressed. Conversely when endogenous Mg-protoporphyrin IX levels are artificially increased by supplementation with the tetrapyrrole precursor, 5-aminolevulinic acid, the expression of nuclear-encoded photosynthetic genes is induced, not repressed. We also demonstrate that NF-treatment leads to a strong down-regulation of tetrapyrrole biosynthesis genes, consistent with the absence of an accumulation of tetrapyrrole intermediates. Finally, there is no correlation between nuclear-gene expression and any of the chlorophyll biosynthetic intermediates over a range of growth conditions and treatments. Instead, it is possible that a perturbation of tetrapyrrole synthesis may lead to localized ROS production or an altered redox state of the plastid, which could mediate retrograde signaling.

Overexpression of chloroplast-targeted ferrochelatase 1 results in a genomes uncoupled chloroplast-to-nucleus retrograde signalling phenotype.

Chloroplast development requires communication between the progenitor plastids and the nucleus, where most of the genes encoding chloroplast proteins reside. Retrograde signals from the chloroplast to the nucleus control the expression of many of these genes, but the signalling pathway is poorly understood. Tetrapyrroles have been strongly implicated as mediators of this signal with the current hypothesis being that haem produced by the activity of ferrochelatase 1 (FC1) is required to promote nuclear gene expression. We have tested this hypothesis by overexpressing FC1 and specifically targeting it to either chloroplasts or mitochondria, two possible locations for this enzyme. Our results show that targeting of FC1 to chloroplasts results in increased expression of the nuclear-encoded chloroplast genes GUN4, CA1, HEMA1, LHCB2.1, CHLH after treatment with Norflurazon (NF) and that this increase correlates to FC1 gene expression and haem production measured by feedback inhibition of protochlorophyllide synthesis. Targeting FC1 to mitochondria did not enhance the expression of nuclear-encoded chloroplast genes after NF treatment. The overexpression of FC1 also increased nuclear gene expression in the absence of NF treatment, demonstrating that this pathway is operational in the absence of a stress treatment. Our results therefore support the hypothesis that haem synthesis is a promotive chloroplast-to-nucleus retrograde signal. However, not all FC1 overexpression lines enhanced nuclear gene expression, suggesting there is still a lot we do not understand about the role of FC1 in this signalling pathway. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Retrograde signals from endosymbiotic organelles: a common control principle in eukaryotic cells.

Endosymbiotic organelles of eukaryotic cells, the plastids, including chloroplasts and mitochondria, are highly integrated into cellular signalling networks. In both heterotrophic and autotrophic organisms, plastids and/or mitochondria require extensive organelle-to-nucleus communication in order to establish a coordinated expression of their own genomes with the nuclear genome, which encodes the majority of the components of these organelles. This goal is achieved by the use of a variety of signals that inform the cell nucleus about the number and developmental status of the organelles and their reaction to changing external environments. Such signals have been identified in both photosynthetic and non-photosynthetic eukaryotes (known as retrograde signalling and retrograde response, respectively) and, therefore, appear to be universal mechanisms acting in eukaryotes of all kingdoms. In particular, chloroplasts and mitochondria both harbour crucial redox reactions that are the basis of eukaryotic life and are, therefore, especially susceptible to stress from the environment, which they signal to the rest of the cell. These signals are crucial for cell survival, lifespan and environmental adjustment, and regulate quality control and targeted degradation of dysfunctional organelles, metabolic adjustments, and developmental signalling, as well as induction of apoptosis. The functional similarities between retrograde signalling pathways in autotrophic and non-autotrophic organisms are striking, suggesting the existence of common principles in signalling mechanisms or similarities in their evolution. Here, we provide a survey for the newcomers to this field of research and discuss the importance of retrograde signalling in the context of eukaryotic evolution. Furthermore, we discuss commonalities and differences in retrograde signalling mechanisms and propose retrograde signalling as a general signalling mechanism in eukaryotic cells that will be also of interest for the specialist. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Light Regulation of Chlorophyll and Glycoalkaloid Biosynthesis During Tuber Greening of Potato S. tuberosum.

Potato, S. tuberosum, is one of the most important global crops, but has high levels of waste due to tuber greening under light, which is associated with the accumulation of neurotoxic glycoalkaloids. However, unlike the situation in de-etiolating seedlings, the mechanisms underlying tuber greening are not well understood. Here, we have investigated the effect of monochromatic blue, red, and far-red light on the regulation of chlorophyll and glycoalkaloid accumulation in potato tubers. Blue and red wavelengths were effective for induction and accumulation of chlorophyll, carotenoids and the two major potato glycoalkaloids, α-solanine and α-chaconine, whereas none of these accumulated in darkness or under far-red light. Key genes in chlorophyll biosynthesis (HEMA1, encoding the rate-limiting enzyme glutamyl-tRNA reductase, GSA, CHLH and GUN4) and six genes (HMG1, SQS, CAS1, SSR2, SGT1 and SGT2) required for glycoalkaloid synthesis were also induced under white, blue, and red light but not in darkness or under far-red light. These data suggest a role for both cryptochrome and phytochrome photoreceptors in chlorophyll and glycoalkaloid accumulation. The contribution of phytochrome was further supported by the observation that far-red light could inhibit white light-induced chlorophyll and glycoalkaloid accumulation and associated gene expression. Transcriptomic analysis of tubers exposed to white, blue, and red light showed that light induction of photosynthesis and tetrapyrrole-related genes grouped into three distinct groups with one group showing a generally progressive induction by light at both 6 h and 24 h, a second group showing induction at 6 h in all light treatments, but induction only by red and white light at 24 h and a third showing just a very moderate light induction at 6 h which was reduced to the dark control level at 24 h. All glycoalkaloid synthesis genes showed a group one profile consistent with what was seen for the most light regulated chlorophyll synthesis genes. Our data provide a molecular framework for developing new approaches to reducing waste due to potato greening.

A Simple Method for Quantification of Protochlorophyllide in Etiolated Arabidopsis Seedlings.

Etiolated seedlings accumulate the chlorophyll biosynthesis intermediate protochlorophyllide (Pchlide) and measuring Pchlide can be important for characterizing photomorphogenic mutants that may be affected in chloroplast development. In this chapter we outline a simple and sensitive method for quantifying Pchlide in extracts of Arabidopsis seedlings using fluorescence spectroscopy. This method can be easily adapted to study chloroplast development in a wide range of plant species.

Diel rhythmicity in amino acid uptake by Prochlorococcus.

The marine cyanobacterium Prochlorococcus, the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light-synchronized axenic Prochlorococcus (PCC9511 strain) culture and (35)S-methionine and (3)H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >10(4) times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G(2) cell cycle stage were consistently 2.2 times higher than those of cells at the G(1) stage. Furthermore, S+G(2) cells upregulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday to 42% at dusk of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations.

Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean.

(35)S-Methionine and (3)H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.

The molecular basis of heme oxygenase deficiency in the pcd1 mutant of pea.

The pcd1 mutant of pea lacks heme oxygenase (HO) activity required for the synthesis of the phytochrome chromophore and is consequently severely deficient in all responses mediated by the phytochrome family of plant photoreceptors. Here we describe the isolation of the gene encoding pea heme oxygenase 1 (PsHO1) and confirm the presence of a mutation in this gene in the pcd1 mutant. PsHO1 shows a high degree of sequence homology to other higher plant HOs, in particular with those from other legume species. Expression of PsHO1 increased in response to white light, but did not respond strongly to narrow band light treatments. Analysis of the biochemical activity of PsHO1 expressed in Escherichia coli demonstrated requirements for reduced ferredoxin, a secondary reductant such as ascorbate and an iron chelator for maximum enzyme activity. Using the crystal structure data from homologous animal and bacterial HOs we have modelled the structure of PsHO1 and demonstrated a high degree of structural conservation despite limited primary sequence homology. However, the catalytic site of PsHO1 is larger than that of animal HOs indicating that it may accommodate an ascorbate molecule in close proximity to the heme. This could provide an explanation for why plant HOs show a strong and saturable dependence on this reductant.

Green or red: what stops the traffic in the tetrapyrrole pathway?

Regulation of tetrapyrrole biosynthesis is crucial to plant metabolism. The two pivotal control points are formation of the initial precursor, 5-aminolaevulinic acid (ALA), and the metal-ion insertion step: chelation of Fe(2+) into protoporphyrin IX leads to haem and phytochromobilin, whereas insertion of Mg(2+) is the first step to chlorophyll. Recent studies with mutants and transgenic plants have demonstrated that perturbation of the branch point affects ALA formation. Moreover, one of the signals that controls the expression of genes for nuclear-encoded chloroplast proteins has been shown to be Mg-protoporphyrin-IX. Here, we discuss the regulation of branch-point flux and the relative contributions of the haem and chlorophyll branches to the regulation of ALA synthesis and thus to flow through the tetrapyrrole pathway.