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.

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.

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'.

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.

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.

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.

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.

Ancestral light and chloroplast regulation form the foundations for C4 gene expression.

C4 photosynthesis acts as a carbon concentrating mechanism that leads to large increases in photosynthetic efficiency. The C4 pathway is found in more than 60 plant lineages1 but the molecular enablers of this evolution are poorly understood. In particular, it is unclear how non-photosynthetic proteins in the ancestral C3 system have repeatedly become strongly expressed and integrated into photosynthesis gene regulatory networks in C4 leaves. Here, we provide clear evidence that in C3 leaves, genes encoding key enzymes of the C4 pathway are already co-regulated with photosynthesis genes and are controlled by both light and chloroplast-to-nucleus signalling. In C4 leaves this regulation becomes increasingly dependent on the chloroplast. We propose that regulation of C4 cycle genes by light and the chloroplast in the ancestral C3 state has facilitated the repeated evolution of the complex and convergent C4 trait.

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.

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.

A model for tetrapyrrole synthesis as the primary mechanism for plastid-to-nucleus signaling during chloroplast biogenesis.

Chloroplast biogenesis involves the co-ordinated expression of the chloroplast and nuclear genomes, requiring information to be sent from the developing chloroplasts to the nucleus. This is achieved through retrograde signaling pathways and can be demonstrated experimentally using the photobleaching herbicide, norflurazon, which in seedlings results in chloroplast damage and the reduced expression of many photosynthesis-related, nuclear genes. Genetic analysis of this pathway points to a major role for tetrapyrrole synthesis in retrograde signaling, as well as a strong interaction with light signaling pathways. Currently, the best model to explain the genetic data is that a specific heme pool generated by flux through ferrochelatase-1 functions as a positive signal to promote the expression of genes required for chloroplast development. We propose that this heme-related signal is the primary positive signal during chloroplast biogenesis, and that treatments and mutations affecting chloroplast transcription, RNA editing, translation, or protein import all impact on the synthesis and/or processing of this signal. A positive signal is consistent with the need to provide information on chloroplast status at all times. We further propose that GUN1 normally serves to restrict the production of the heme signal. In addition to a positive signal re-enforcing chloroplast development under normal conditions, aberrant chloroplast development may produce a negative signal due to accumulation of unbound chlorophyll biosynthesis intermediates, such as Mg-porphyrins. Under these conditions a rapid shut-down of tetrapyrrole synthesis is required. We propose that accumulation of these intermediates results in a rapid light-dependent inhibition of nuclear gene expression that is most likely mediated via singlet oxygen generated by photo-excitation of Mg-porphyrins. Thus, the tetrapyrrole pathway may provide both positive and inhibitory signals to control expression of nuclear genes.

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.

Improving photosynthesis for algal biofuels: toward a green revolution.

Biofuels derived from marine algae are a potential source of sustainable energy that can contribute to future global demands. The realisation of this potential will require manipulation of the fundamental biology of algal physiology to increase the efficiency with which solar energy is ultimately converted into usable biomass. This 'photosynthetic solar energy conversion efficiency' sets an upper limit on the potential of algal-derived biofuels. In this review, we outline photosynthetic molecular targets that could be manipulated to increase the efficiency and yield of algal biofuel production. We also highlight modern 'omic' and high-throughput technologies that might enable identification, selection and improvement of algal cell lines on timescales relevant for achieving significant contributions to future energy solutions.

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.

The cell biology of tetrapyrroles: a life and death struggle.

Tetrapyrroles such as chlorophyll and heme are co-factors for essential proteins involved in a wide variety of crucial cellular functions. Nearly 2% of the proteins encoded by the Arabidopsis thaliana genome are thought to bind tetrapyrroles, demonstrating their central role in plant metabolism. Although the enzymes required for tetrapyrrole biosynthesis are well characterized, there are still major questions about the regulation of the pathway, and the transport of tetrapyrroles within cells. These issues are important, as misregulation of tetrapyrrole metabolism can lead to severe photo-oxidative stress, and because tetrapyrroles have been implicated in signaling pathways coordinating interactions between plant organelles. In this review, we discuss the cell biology of tetrapyrrole metabolism and its implications for tetrapyrroles as signaling molecules.

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.

Light signalling pathways regulating the Mg-chelatase branchpoint of chlorophyll synthesis during de-etiolation in Arabidopsis thaliana.

Precise regulation of tetrapyrrole synthesis is critical for plant survival when seedlings first emerge into the light. At this time there is a massive increase in demand for chlorophyll to drive the assembly of the photosynthetic apparatus. To understand how this demand is met we have followed the expression of genes encoding the chelatase enzymes at the branchpoint between chlorophyll and heme synthesis. Dark-grown Arabidopsis thaliana seedlings were transferred to continuous white, red, far-red or blue light and the expression of eight tetrapyrrole pathway genes was followed using real-time RT-PCR. Our results show that the CHLH gene encoding the H subunit of Mg-chelatase was induced by light under all conditions with an initial peak after 2-4 h light. The other Mg-chelatase subunit genes CHLI and CHLD and the ferrochelatase genes FC1 and FC2 were not strongly regulated at the level of transcript abundance, but the Mg-chelatase regulator GUN4 had an expression profile almost identical to that observed for CHLH. The CHLM gene encoding Mg-protoporphyrin IX methyltransferase, the next enzyme in the pathway, was also light regulated, but showed a very different pattern of expression. Using photoreceptor mutants it was demonstrated that regulation of CHLH and GUN4 is primarily under the control of phytochromes A and B with some input from the cryptochromes. Induction of CHLH and GUN4 under red and far-red light was also compromised in the phytochrome-signalling mutants, fhy1 and fhy3. These results establish GUN4 as a major target of photoreceptor regulation during the earliest stages of de-etiolation.