Cadmium interference with iron sensing reveals transcriptional programs sensitive and insensitive to reactive oxygen species.

Iron (Fe) is an essential micronutrient whose uptake is tightly regulated to prevent either deficiency or toxicity. Cadmium (Cd) is a non-essential element that induces both Fe deficiency and toxicity; however, the mechanisms behind these Fe/Cd-induced responses are still elusive. Here we explored Cd- and Fe-associated responses in wild-type Arabidopsis and in a mutant that overaccumulates Fe (opt3-2). Gene expression profiling revealed a large overlap between transcripts induced by Fe deficiency and Cd exposure. Interestingly, the use of opt3-2 allowed us to identify additional gene clusters originally induced by Cd in the wild type but repressed in the opt3-2 background. Based on the high levels of H2O2 found in opt3-2, we propose a model where reactive oxygen species prevent the induction of genes that are induced in the wild type by either Fe deficiency or Cd. Interestingly, a defined cluster of Fe-responsive genes was found to be insensitive to this negative feedback, suggesting that their induction by Cd is more likely to be the result of an impaired Fe sensing. Overall, our data suggest that Fe deficiency responses are governed by multiple inputs and that a hierarchical regulation of Fe homeostasis prevents the induction of specific networks when Fe and H2O2 levels are elevated.

Loss of inner-envelope K+/H+ exchangers impairs plastid rRNA maturation and gene expression.

The inner-envelope K+ EFFLUX ANTIPORTERS (KEA) 1 and 2 are critical for chloroplast development, ion homeostasis, and photosynthesis. However, the mechanisms by which changes in ion flux across the envelope affect organelle biogenesis remained elusive. Chloroplast development requires intricate coordination between the nuclear genome and the plastome. Many mutants compromised in plastid gene expression (PGE) display a virescent phenotype, that is delayed greening. The phenotypic appearance of Arabidopsis thaliana kea1 kea2 double mutants fulfills this criterion, yet a link to PGE has not been explored. Here, we show that a simultaneous loss of KEA1 and KEA2 results in maturation defects of the plastid ribosomal RNAs. This may be caused by secondary structure changes of rRNA transcripts and concomitant reduced binding of RNA-processing proteins, which we documented in the presence of skewed ion homeostasis in kea1 kea2. Consequently, protein synthesis and steady-state levels of plastome-encoded proteins remain low in mutants. Disturbance in PGE and other signs of plastid malfunction activate GENOMES UNCOUPLED 1-dependent retrograde signaling in kea1 kea2, resulting in a dramatic downregulation of GOLDEN2-LIKE transcription factors to halt expression of photosynthesis-associated nuclear-encoded genes (PhANGs). PhANG suppression delays the development of fully photosynthesizing kea1 kea2 chloroplasts, probably to avoid progressing photo-oxidative damage. Overall, our results reveal that KEA1/KEA2 function impacts plastid development via effects on RNA-metabolism and PGE.

Stromal NADH supplied by PHOSPHOGLYCERATE DEHYDROGENASE3 is crucial for photosynthetic performance.

During photosynthesis, electrons travel from light-excited chlorophyll molecules along the electron transport chain to the final electron acceptor nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH, which fuels the Calvin-Benson-Bassham cycle (CBBC). To allow photosynthetic reactions to occur flawlessly, a constant resupply of the acceptor NADP is mandatory. Several known stromal mechanisms aid in balancing the redox poise, but none of them utilizes the structurally highly similar coenzyme NAD(H). Using Arabidopsis (Arabidopsis thaliana) as a C3-model, we describe a pathway that employs the stromal enzyme PHOSPHOGLYCERATE DEHYDROGENASE 3 (PGDH3). We showed that PGDH3 exerts high NAD(H)-specificity and is active in photosynthesizing chloroplasts. PGDH3 withdrew its substrate 3-PGA directly from the CBBC. As a result, electrons become diverted from NADPH via the CBBC into the separate NADH redox pool. pgdh3 loss-of-function mutants revealed an overreduced NADP(H) redox pool but a more oxidized plastid NAD(H) pool compared to wild-type plants. As a result, photosystem I acceptor side limitation increased in pgdh3. Furthermore, pgdh3 plants displayed delayed CBBC activation, changes in nonphotochemical quenching, and altered proton motive force partitioning. Our fluctuating light-stress phenotyping data showed progressing photosystem II damage in pgdh3 mutants, emphasizing the significance of PGDH3 for plant performance under natural light environments. In summary, this study reveals an NAD(H)-specific mechanism in the stroma that aids in balancing the chloroplast redox poise. Consequently, the stromal NAD(H) pool may provide a promising target to manipulate plant photosynthesis.

Plastocyanin is the long-range electron carrier between photosystem II and photosystem I in plants.

In photosynthetic electron transport, large multiprotein complexes are connected by small diffusible electron carriers, the mobility of which is challenged by macromolecular crowding. For thylakoid membranes of higher plants, a long-standing question has been which of the two mobile electron carriers, plastoquinone or plastocyanin, mediates electron transport from stacked grana thylakoids where photosystem II (PSII) is localized to distant unstacked regions of the thylakoids that harbor PSI. Here, we confirm that plastocyanin is the long-range electron carrier by employing mutants with different grana diameters. Furthermore, our results explain why higher plants have a narrow range of grana diameters since a larger diffusion distance for plastocyanin would jeopardize the efficiency of electron transport. In the light of recent findings that the lumen of thylakoids, which forms the diffusion space of plastocyanin, undergoes dynamic swelling/shrinkage, this study demonstrates that plastocyanin diffusion is a crucial regulatory element of plant photosynthetic electron transport.

A proteoliposome-based system reveals how lipids control photosynthetic light harvesting.

Integral membrane proteins are exposed to a complex and dynamic lipid environment modulated by nonbilayer lipids that can influence protein functions by lipid-protein interactions. The nonbilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant lipid in plant photosynthetic thylakoid membranes, but its impact on the functionality of energy-converting membrane protein complexes is unknown. Here, we optimized a detergent-based reconstitution protocol to develop a proteoliposome technique that incorporates the major light-harvesting complex II (LHCII) into compositionally well-defined large unilamellar lipid bilayer vesicles to study the impact of MGDG on light harvesting by LHCII. Using steady-state fluorescence spectroscopy, CD spectroscopy, and time-correlated single-photon counting, we found that both chlorophyll fluorescence quantum yields and fluorescence lifetimes clearly indicate that the presence of MGDG in lipid bilayers switches LHCII from a light-harvesting to a more energy-quenching mode that dissipates harvested light into heat. It is hypothesized that in the in vitro system developed here, MGDG controls light harvesting of LHCII by modulating the hydrostatic lateral membrane pressure profile in the lipid bilayer sensed by LHCII-bound peripheral pigments.

Photosynthesis in Arabidopsis Is Unaffected by the Function of the Vacuolar K+ Channel TPK3.

Photosynthesis is limited by the slow relaxation of nonphotochemical quenching, which primarily dissipates excess absorbed light energy as heat. Because the heat dissipation process is proportional to light-driven thylakoid lumen acidification, manipulating thylakoid ion and proton flux via transport proteins could improve photosynthesis. However, an important aspect of the current understanding of the thylakoid ion transportome is inaccurate. Using fluorescent protein fusions, we show that the Arabidopsis (Arabidopsis thaliana) two-pore K+ channel TPK3, which had been reported to mediate thylakoid K+ flux, localizes to the tonoplast, not the thylakoid. The localization of TPK3 outside of the thylakoids is further supported by the absence of TPK3 in isolated thylakoids as well as the inability of isolated chloroplasts to import TPK3 protein. In line with the subcellular localization of TPK3 in the vacuole, we observed that photosynthesis in the Arabidopsis null mutant tpk3-1, which carries a transfer DNA insertion in the first exon, remains unaffected. To gain a comprehensive understanding of how thylakoid ion flux impacts photosynthetic efficiency under dynamic growth light regimes, we performed long-term photosynthesis imaging of established and newly isolated transthylakoid K+- and Cl--flux mutants. Our results underpin the importance of the thylakoid ion transport proteins potassium cation efflux antiporter KEA3 and voltage-dependent chloride channel VCCN1 and suggest that the activity of yet unknown K+ channel(s), but not TPK3, is critical for optimal photosynthesis in dynamic light environments.

Reduced Arogenate Dehydratase Expression: Ramifications for Photosynthesis and Metabolism.

Arogenate dehydratase (ADT) catalyzes the final step of phenylalanine (Phe) biosynthesis. Previous work showed that ADT-deficient Arabidopsis (Arabidopsis thaliana) mutants had significantly reduced lignin contents, with stronger reductions in lines that had deficiencies in more ADT isoforms. Here, by analyzing Arabidopsis ADT mutants using our phenomics facility and ultra-performance liquid chromatography-mass spectrometry-based metabolomics, we describe the effects of the modulation of ADT on photosynthetic parameters and secondary metabolism. Our data indicate that a reduced carbon flux into Phe biosynthesis in ADT mutants impairs the consumption of photosynthetically produced ATP, leading to an increased ATP/ADP ratio, the overaccumulation of transitory starch, and lower electron transport rates. The effect on electron transport rates is caused by an increase in proton motive force across the thylakoid membrane that down-regulates photosystem II activity by the high-energy quenching mechanism. Furthermore, quantitation of secondary metabolites in ADT mutants revealed reduced flavonoid, phenylpropanoid, lignan, and glucosinolate contents, including glucosinolates that are not derived from aromatic amino acids, and significantly increased contents of putative galactolipids and apocarotenoids. Additionally, we used real-time atmospheric monitoring mass spectrometry to compare respiration and carbon fixation rates between the wild type and adt3/4/5/6, our most extreme ADT knockout mutant, which revealed no significant difference in both night- and day-adapted plants. Overall, these data reveal the profound effects of altered ADT activity and Phe metabolism on secondary metabolites and photosynthesis with implications for plant improvement.

Proton Gradients and Proton-Dependent Transport Processes in the Chloroplast.

Proton gradients are fundamental to chloroplast function. Across thylakoid membranes, the light induced -proton gradient is essential for ATP synthesis. As a result of proton pumping into the thylakoid lumen, an alkaline stromal pH develops, which is required for full activation of pH-dependent Calvin Benson cycle enzymes. This implies that a pH gradient between the cytosol (pH 7) and the stroma (pH 8) is established upon illumination. To maintain this pH gradient chloroplasts actively extrude protons. More than 30 years ago it was already established that these proton fluxes are electrically counterbalanced by Mg(2+), K(+), or Cl(-) fluxes, but only recently the first transport systems that regulate the pH gradient were identified. Notably several (Na(+),K(+))/H(+) antiporter systems where identified, that play a role in pH gradient regulation, ion homeostasis, osmoregulation, or coupling of secondary active transport. The established pH gradients are important to drive uptake of essential ions and solutes, but not many transporters involved have been identified to date. In this mini review we summarize the current status in the field and the open questions that need to be addressed in order to understand how pH gradients are maintained, how this is interconnected with other transport processes and what this means for chloroplast function.

Lack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth.

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO2 is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by ¹4N/¹5N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in ß-oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat.

The metabolic status drives acclimation of iron deficiency responses in Chlamydomonas reinhardtii as revealed by proteomics based hierarchical clustering and reverse genetics.

Iron is a crucial cofactor in numerous redox-active proteins operating in bioenergetic pathways including respiration and photosynthesis. Cellular iron management is essential to sustain sufficient energy production and minimize oxidative stress. To produce energy for cell growth, the green alga Chlamydomonas reinhardtii possesses the metabolic flexibility to use light and/or carbon sources such as acetate. To investigate the interplay between the iron-deficiency response and growth requirements under distinct trophic conditions, we took a quantitative proteomics approach coupled to innovative hierarchical clustering using different "distance-linkage combinations" and random noise injection. Protein co-expression analyses of the combined data sets revealed insights into cellular responses governing acclimation to iron deprivation and regulation associated with photosynthesis dependent growth. Photoautotrophic growth requirements as well as the iron deficiency induced specific metabolic enzymes and stress related proteins, and yet differences in the set of induced enzymes, proteases, and redox-related polypeptides were evident, implying the establishment of distinct response networks under the different conditions. Moreover, our data clearly support the notion that the iron deficiency response includes a hierarchy for iron allocation within organelles in C. reinhardtii. Importantly, deletion of a bifunctional alcohol and acetaldehyde dehydrogenase (ADH1), which is induced under low iron based on the proteomic data, attenuates the remodeling of the photosynthetic machinery in response to iron deficiency, and at the same time stimulates expression of stress-related proteins such as NDA2, LHCSR3, and PGRL1. This finding provides evidence that the coordinated regulation of bioenergetics pathways and iron deficiency response is sensitive to the cellular and chloroplast metabolic and/or redox status, consistent with systems approach data.

Concerted action of the new Genomic Peptide Finder and AUGUSTUS allows for automated proteogenomic annotation of the Chlamydomonas reinhardtii genome.

The use and development of post-genomic tools naturally depends on large-scale genome sequencing projects. The usefulness of post-genomic applications is dependent on the accuracy of genome annotations, for which the correct identification of intron-exon borders in complex genomes of eukaryotic organisms is often an error-prone task. Although automated algorithms for predicting intron-exon structures are available, supporting exon evidence is necessary to achieve comprehensive genome annotation. Besides cDNA and EST support, peptides identified via MS/MS can be used as extrinsic evidence in a proteogenomic approach. We describe an improved version of the Genomic Peptide Finder (GPF), which aligns de novo predicted amino acid sequences to the genomic DNA sequence of an organism while correcting for peptide sequencing errors and accounting for the possibility of splicing. We have coupled GPF and the gene finding program AUGUSTUS in a way that provides automatic structural annotations of the Chlamydomonas reinhardtii genome, using highly unbiased GPF evidence. A comparison of the AUGUSTUS gene set incorporating GPF evidence to the standard JGI FM4 (Filtered Models 4) gene set reveals 932 GPF peptides that are not contained in the Filtered Models 4 gene set. Furthermore, the GPF evidence improved the AUGUSTUS gene models by altering 65 gene models and adding three previously unidentified genes.