Life on earth is dependent on the photosynthetic conversion of light energy into chemical energy. However, absorption of excess sunlight can damage the photosynthetic machinery and limit photosynthetic activity, thereby affecting growth and productivity. Photosynthetic light harvesting can be down-regulated by nonphotochemical quenching (NPQ). A major component of NPQ is qE (energy-dependent nonphotochemical quenching), which allows dissipation of light energy as heat. Photodamage peaks in the UV-B part of the spectrum, but whether and how UV-B induces qE are unknown. Plants are responsive to UV-B via the UVR8 photoreceptor. Here, we report in the green alga Chlamydomonas reinhardtii that UVR8 induces accumulation of specific members of the light-harvesting complex (LHC) superfamily that contribute to qE, in particular LHC Stress-Related 1 (LHCSR1) and Photosystem II Subunit S (PSBS). The capacity for qE is strongly induced by UV-B, although the patterns of qE-related proteins accumulating in response to UV-B or to high light are clearly different. The competence for qE induced by acclimation to UV-B markedly contributes to photoprotection upon subsequent exposure to high light. Our study reveals an anterograde link between photoreceptor-mediated signaling in the nucleocytosolic compartment and the photoprotective regulation of photosynthetic activity in the chloroplast.L ight is essential for photosynthesis, but absorption of excess light energy is detrimental. To avoid photodamage, photosynthetic light harvesting is regulated by nonphotochemical quenching (NPQ), which allows dissipation of harmful excess energy as heat through its qE (energy-dependent nonphotochemical quenching) component (1-6). Specialized members of the light harvesting complex (LHC) protein family, such as Photosystem II Subunit S (PSBS) in higher plants or members of the LHC Stress-Related (LHCSR) family in mosses and algae, are central to qE (7-11). Protonation of key residues in these proteins triggers qE in response to the acidification of the thylakoid lumen, which is coupled to photosynthetic electron transport (7, 9). Furthermore, the deepoxidation of violaxanthin to zeaxanthin, which is also activated by the acidification of the thylakoid lumen, enhances qE (12). In response to high levels of visible light, LHCSR3 protein accumulation is of major importance for qE capacity in Chlamydomonas reinhardtii (11). The induction of LHCSR3 expression under high light is thought to involve retrograde signaling, from the chloroplast to nuclear gene expression (13), and recent data show that the response is also dependent on the phototropin (PHOT) blue light photoreceptor (14).UV-B radiation is intrinsic to sunlight reaching the earth surface and is potentially damaging to living tissues. UV-B stress tolerance is induced through the specific activation of acclimation responses (15)(16)(17)(18)(19)(20). Plants sense UV-B radiation via the homodimeric UV-B photoreceptor UV Resistance Locus 8 (UVR8) (21-23) that is mainly localized in the cytosol ...

We have identified a yeast nuclear gene (FMC1) that is required at elevated temperatures (37°C) for the formation/stability of the F 1 sector of the mitochondrial ATP synthase. Western blot analysis showed that Fmc1p is a soluble protein located in the mitochondrial matrix. At elevated temperatures in yeast cells lacking Fmc1p, the ␣-F 1 and ␤-F 1 proteins are synthesized, transported, and processed to their mature size. However, instead of being incorporated into a functional F 1 oligomer, they form large aggregates in the mitochondrial matrix. Identical perturbations were reported previously for yeast cells lacking either Atp12p or Atp11p, two specific assembly factors of the F 1 sector (Ackerman, S. H., and Tzagoloff, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4986 -4990), and we show that the absence of Fmc1p can be efficiently compensated for by increasing the expression of Atp12p. However, unlike Atp12p and Atp11p, Fmc1p is not required in normal growth conditions (28 -30°C). We propose that Fmc1p is required for the proper folding/stability or functioning of Atp12p in heat stress conditions. F 1 F o -ATP synthases play a major role in cellular energy production. They are found in the plasma membranes of bacteria, thylakoid membranes of chloroplasts, and in the inner membrane of mitochondria. They use a proton gradient across their host membrane to produce ATP from ADP and inorganic phosphate (1, 2). This enzyme contains two distinct parts, called F o and F 1 . The F o mediates the transmembrane transport of protons, and the synthesis of ATP takes place on the F 1 .The F 1 contains five different types of subunits in the stoichiometric ratio ␣ 3 ␤ 3 ␥␦⑀ (3, 4). The three-dimensional structures of F 1 from bovine heart (5), rat liver (6) and yeast (7) show that the ␣-and ␤-subunits alternate in a hexagonal array with a central cavity occupied by the amino and carboxyl termini of the ␥-subunit. The interfaces between the ␣-and ␤-subunits form three catalytic and three noncatalytic nucleotide binding sites.In the yeast Saccharomyces cerevisiae, the F 1 subunits are encoded in the nucleus (8 -12), synthesized in the cytoplasm, imported into mitochondria as unfolded polypeptide chains (13), and then folded in the mitochondrial matrix with the help of Hsp60p and Hsp10p (14). The oligomerization of the F 1 monomers is assisted by two proteins called Atp12p and Atp11p. These interact directly with the ␣-F 1 and ␤-F 1 proteins, respectively (15, 16). In yeast strains lacking either Atp11p or Atp12p, the ␣-F 1 and ␤-F 1 proteins aggregate in the mitochondrial matrix (17). Thus it is believed that Atp12p and Atp11p facilitate the formation of ␣␤ heterodimers by protecting these two F 1 subunits from non-productive interactions (16).We report in this study the identification of Fmc1p, a novel protein required for the formation or stability of the F 1 oligomer. Like Atp11p and Atp12p, its absence also results in the aggregation of the ␣-F 1 and ␤-F 1 proteins. However, this is seen only at elevated temperatures (37°...

Suberin is a hydrophobic biopolymer that can be deposited at the periphery of cells, forming protective barriers against biotic and abiotic stress. In roots, suberin forms lamellae at the periphery of endodermal cells where it plays crucial roles in the control of water and mineral transport. Suberin formation is highly regulated by developmental and environmental cues. However, the mechanisms controlling its spatiotemporal regulation are poorly understood. Here, we show that endodermal suberin is regulated independently by developmental and exogenous signals to fine-tune suberin deposition in roots. We found a set of four MYB transcription factors (MYB41, MYB53, MYB92, and MYB93), each of which is individually regulated by these two signals and is sufficient to promote endodermal suberin. Mutation of these four transcription factors simultaneously through genome editing leads to a dramatic reduction in suberin formation in response to both developmental and environmental signals. Most suberin mutants analyzed at physiological levels are also affected in another endodermal barrier made of lignin (Casparian strips) through a compensatory mechanism. Through the functional analysis of these four MYBs, we generated plants allowing unbiased investigation of endodermal suberin function, without accounting for confounding effects due to Casparian strip defects, and were able to unravel specific roles of suberin in nutrient homeostasis.

SummaryChloroplast transformation in microalgae offers great promise for the production of proteins of pharmaceutical interest or for the development of novel biofuels. For many applications, high level expression of transgenes is desirable. We have transformed the chloroplast of Chlamydomonas reinhardtii with two genes, acrV and vapA, which encode antigens from the fish pathogen Aeromonas salmonicida. The promoters and 5¢ untranslated regions of four chloroplast genes were compared for their ability to drive expression of the bacterial genes. The highest levels of expression were obtained when they were placed under the control of the cis-acting elements from the psaA-exon1 gene. The expression of these chimeric genes was further increased when a nuclear mutation that affects a factor involved in psaA splicing was introduced in the genetic background of the chloroplast transformants. Accumulation of both the chimeric mRNAs and the recombinant proteins was dramatically increased, indicating that negative feedback loops limit the expression of chloroplast transgenes. Our results demonstrate the potential of manipulating anterograde signalling to alter negative regulatory feedback loops in the chloroplast and improve transgene expression.

The F 1 component of mitochondrial ATP synthase is an oligomeric assembly of five different subunits, ␣, ␤, ␥, ␦, and ⑀. In terms of mass, the bulk of the structure (ϳ90%) is provided by the ␣ and ␤ subunits, which form an (␣ ␤) 3 hexamer with adenine nucleotide binding sites at the ␣/␤ interfaces. We report here ultrastructural and immunocytochemical analyses of yeast mutants that are unable to form the ␣ 3 ␤ 3 oligomer, either because the ␣ or the ␤ subunit is missing or because the cells are deficient for proteins that mediate F 1 assembly (e.g. Atp11p, Atp12p, or Fmc1p). The F 1 ␣ and ␤ subunits of such mutant strains are detected within large electron-dense particles in the mitochondrial matrix. The composition of the aggregated species is principally full-length F 1 ␣ and/or ␤ subunit protein that has been processed to remove the amino-terminal targeting peptide. To our knowledge this is the first demonstration of mitochondrial inclusion bodies that are formed largely of one particular protein species. We also show that yeast mutants lacking the ␣ 3 ␤ 3 oligomer are devoid of mitochondrial cristae and are severely deficient for respiratory complexes III and IV. These observations are in accord with other studies in the literature that have pointed to a central role for the ATP synthase in biogenesis of the mitochondrial inner membrane.

Iron (Fe) is a major micronutrient and is required for plant growth and development. Nongrass species have evolved a reduction-based strategy to solubilize and take up Fe. The secretion of Fe-mobilizing coumarins (e.g. fraxetin, esculetin and sideretin) by plant roots plays an important role in this process. Although the biochemical mechanisms leading to their biosynthesis have been well described, very little is known about their cellular and subcellular localization or their mobility within plant tissues. Spectral imaging was used to monitor, in Arabidopsis thaliana, the in planta localization of Fe-mobilizing coumarins and scopolin. Molecular, genetic and biochemical approaches were also used to investigate the dynamics of coumarin accumulation in roots. These approaches showed that root hairs play a major role in scopoletin secretion, whereas fraxetin and esculetin secretion occurs through all epidermis cells. The findings of this study also showed that the transport of coumarins from the cortex to the rhizosphere relies on the PDR9 transporter under Fe-deficient conditions. Additional experiments support the idea that coumarins move throughout the plant body via the xylem sap and that several plant species can take up coumarins present in the surrounding media. Altogether, the data presented here demonstrate that coumarin storage and accumulation in roots is a highly complex and dynamic process.

Many chloroplast transcripts are protected against exonucleolytic degradation by RNA-binding proteins. Such interactions can lead to the accumulation of short RNAs (sRNAs) that represent footprints of the protein partner. By mining existing data sets of Chlamydomonas reinhardtii small RNAs, we identify chloroplast sRNAs. Two of these correspond to the 5′-ends of the mature psbB and psbH messenger RNAs (mRNAs), which are both stabilized by the nucleus-encoded protein Mbb1, a member of the tetratricopeptide repeat family. Accordingly, we find that the two sRNAs are absent from the mbb1 mutant. Using chloroplast transformation and site-directed mutagenesis to survey the psbB 5′ UTR, we identify a cis-acting element that is essential for mRNA accumulation. This sequence is also found in the 5′ UTR of psbH, where it plays a role in RNA processing. The two sRNAs are centered on these cis-acting elements. Furthermore, RNA binding assays in vitro show that Mbb1 associates with the two elements specifically. Taken together, our data identify a conserved cis-acting element at the extremity of the psbH and psbB 5′ UTRs that plays a role in the processing and stability of the respective mRNAs through interactions with the tetratricopeptide repeat protein Mbb1 and leads to the accumulation of protected sRNAs.

In a previous study we have identified Fmc1p, a mitochondrial protein involved in the assembly/stability of the yeast F 0 F 1 -ATP synthase at elevated temperatures. The ⌬fmc1 mutant was shown to exhibit a severe phenotype of very slow growth on respiratory substrates at 37°C. We have isolated ODC1 as a multicopy suppressor of the fmc1 deletion restoring a good respiratory growth. Odc1p expression level was estimated to be at least 10 times higher in mitochondria isolated from the ⌬fmc1/ODC1 transformant as compared with wild type mitochondria. Interestingly, ODC1 encodes an oxodicarboxylate carrier, which transports ␣-ketoglutarate and ␣-ketoadipate or any other transported tricarboxylic acid cycle intermediate in a counter-exchange through the inner mitochondrial membrane. We show that the suppression of the respiratory-growth-deficient fmc1 by the overexpressed Odc1p was not due to a restored stable ATP synthase. Instead, the rescuing mechanism involves an increase in the flux of tricarboxylic acid cycle intermediate from the cytosol into the mitochondria, leading to an increase in the ␣-ketoglutarate oxidative decarboxylation, resulting in an increase in mitochondrial substrate-level-dependent ATP synthesis. This mechanism of metabolic bypass of a defective ATP synthase unravels the physiological importance of intramitochondrial substrate-level phosphorylations. This unexpected result might be of interest for the development of therapeutic solutions in pathologies associated with defects in the oxidative phosphorylation system.In the inner mitochondrial membrane, the F 0 F 1 -ATP synthase performs the late step of the oxidative phosphorylations. This hetero-oligomer uses the electrochemical transmembrane proton gradient generated by the respiratory chain to catalyze ATP synthesis from ADP and inorganic phosphate (for reviews, see Refs. 1 and 2). It is composed of two distinct domains, a membrane-integrated F 0 domain containing a proton channel and a hydrophilic peripheral F 1 domain bearing the catalytic sites for ATP synthesis.This enzyme comprises about 20 different subunits for an overall molecular mass approaching 600 kDa. The genes encoding these proteins are located part in the nucleus and part in the mitochondrion itself. The subunits of nuclear origin are synthesized in the cytoplasm and then imported into the mitochondrion (for review, see Ref.3), whereas the mitochondrial DNA-encoded subunits are synthesized within the mitochondrion. This genetic compartmentalization makes the assembly of the ATP synthase complex a particularly intricate process. Studies in Saccharomyces cerevisiae have shown that specific proteins, usually called assembly factors, not belonging to the final complex, are required in the different steps of the enzyme biogenesis. Three such proteins, Atp11p, Atp12p, and Fmc1p, were shown to facilitate the assembly of the ATP synthase F 1 component (4 -6), which is made of five different nuclear-encoded proteins in the ␣ 3 ␤ 3 ␥␦⑀ stoichiometry (7). The ␣ and ␤ subunits alternate in ...