A proxitome-RNA-capture approach reveals that processing bodies repress coregulated hub genes.

Cellular condensates are usually ribonucleoprotein assemblies with liquid- or solid-like properties. Because these subcellular structures lack a delineating membrane, determining their compositions is difficult. Here we describe a proximity-biotinylation approach for capturing the RNAs of the condensates known as processing bodies (PBs) in Arabidopsis (Arabidopsis thaliana). By combining this approach with RNA detection, in silico, and high-resolution imaging approaches, we studied PBs under normal conditions and heat stress. PBs showed a much more dynamic RNA composition than the total transcriptome. RNAs involved in cell wall development and regeneration, plant hormonal signaling, secondary metabolism/defense, and RNA metabolism were enriched in PBs. RNA-binding proteins and the liquidity of PBs modulated RNA recruitment, while RNAs were frequently recruited together with their encoded proteins. In PBs, RNAs follow distinct fates: in small liquid-like PBs, RNAs get degraded while in more solid-like larger ones, they are stored. PB properties can be regulated by the actin-polymerizing SCAR (suppressor of the cyclic AMP)-WAVE (WASP family verprolin homologous) complex. SCAR/WAVE modulates the shuttling of RNAs between PBs and the translational machinery, thereby adjusting ethylene signaling. In summary, we provide an approach to identify RNAs in condensates that allowed us to reveal a mechanism for regulating RNA fate.

Structural basis of metabolite transport by the chloroplast outer envelope channel OEP21.

Triose phosphates (TPs) are the primary products of photosynthetic CO2 fixation in chloroplasts, which need to be exported into the cytosol across the chloroplast inner envelope (IE) and outer envelope (OE) membranes to sustain plant growth. While transport across the IE is well understood, the mode of action of the transporters in the OE remains unclear. Here we present the high-resolution nuclear magnetic resonance (NMR) structure of the outer envelope protein 21 (OEP21) from garden pea, the main exit pore for TPs in C3 plants. OEP21 is a cone-shaped ß-barrel pore with a highly positively charged interior that enables binding and translocation of negatively charged metabolites in a competitive manner, up to a size of ~1 kDa. ATP stabilizes the channel and keeps it in an open state. Despite the broad substrate selectivity of OEP21, these results suggest that control of metabolite transport across the OE might be possible.

Two wrongs make a right: heat stress reversion of a male-sterile Brassica napus line.

Male-sterile lines play important roles in plant breeding to obtain hybrid vigour. The male sterility Lembke (MSL) system is a thermosensitive genic male sterility system of Brassica napus and is one of the main systems used in European rapeseed breeding. Interestingly, the MSL system shows high similarity to the 9012AB breeding system from China, including the ability to revert to fertile in high temperature conditions. Here we demonstrate that the MSL system is regulated by the same restorer of fertility gene BnaC9-Tic40 as the 9012AB system, which is related to the translocon at the inner envelope membrane of chloroplasts 40 (TIC40) from Arabidopsis. The male sterility gene of the MSL system was also identified to encode a chloroplast-localized protein which we call BnChimera; this gene shows high sequence similarity to the sterility gene previously described for the 9012AB system. For the first time, a direct protein interaction between BnaC9-Tic40 and the BnChimera could be demonstrated. In addition, we identify the corresponding amino acids that mediate this interaction and suggest how BnaC9-Tic40 acts as the restorer of fertility. Using an RNA-seq approach, the effects of heat treatment on the male fertility restoration of the C545 MSL system line were investigated. These data demonstrate that many pollen developmental pathways are affected by higher temperatures. It is hypothesized that heat stress reverses the male sterility via a combination of slower production of cell wall precursors in plastids and a slower flower development, which ultimately results in fertile pollen. The potential breeding applications of these results are discussed regarding the use of the MSL system in producing thermotolerant fertile plants.

Two plastid POLLUX ion channel-like proteins are required for stress-triggered stromal Ca2+release.

Two decades ago, large cation currents were discovered in the envelope membranes of Pisum sativum L. (pea) chloroplasts. The deduced K+-permeable channel was coined fast-activating chloroplast cation channel but its molecular identity remained elusive. To reveal candidates, we mined proteomic datasets of isolated pea envelopes. Our search uncovered distant members of the nuclear POLLUX ion channel family. Since pea is not amenable to molecular genetics, we used Arabidopsis thaliana to characterize the two gene homologs. Using several independent approaches, we show that both candidates localize to the chloroplast envelope membrane. The proteins, designated PLASTID ENVELOPE ION CHANNELS (PEC1/2), form oligomers with regulator of K+ conductance domains protruding into the intermembrane space. Heterologous expression of PEC1/2 rescues yeast mutants deficient in K+ uptake. Nuclear POLLUX ion channels cofunction with Ca2+ channels to generate Ca2+ signals, critical for establishing mycorrhizal symbiosis and root development. Chloroplasts also exhibit Ca2+ transients in the stroma, probably to relay abiotic and biotic cues between plastids and the nucleus via the cytosol. Our results show that pec1pec2 loss-of-function double mutants fail to trigger the characteristic stromal Ca2+ release observed in wild-type plants exposed to external stress stimuli. Besides this molecular abnormality, pec1pec2 double mutants do not show obvious phenotypes. Future studies of PEC proteins will help to decipher the plant's stress-related Ca2+ signaling network and the role of plastids. More importantly, the discovery of PECs in the envelope membrane is another critical step towards completing the chloroplast ion transport protein inventory.

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.

The topology of plastid inner envelope potassium cation efflux antiporter KEA1 provides new insights into its regulatory features.

The plastid potassium cation efflux antiporters (KEAs) are important for chloroplast function, development, and photosynthesis. To understand their regulation at the protein level is therefore of fundamental importance. Prior studies have focused on the regulatory K+ transport and NAD-binding (KTN) domain in the C-terminus of the thylakoid carrier KEA3 but the localization of this domain remains unclear. While all three plastid KEA members are highly conserved in their transmembrane region and the C-terminal KTN domain, only the inner envelope KEA family members KEA1 and KEA2 carry a long soluble N-terminus. Interestingly, this region is acetylated at lysine 168 by the stromal acetyltransferase enzyme NSI. If an odd number of transmembrane domains existed for inner envelope KEAs, as it was suggested for all three plastid KEA carriers, regulatory domains and consequently protein regulation would occur on opposing sides of the inner envelope. In this study we therefore set out to investigate the topology of inner envelope KEA proteins. Using a newly designed antibody specific to the envelope KEA1 N-terminus and transgenic Arabidopsis plants expressing a C-terminal KEA1-YFP fusion protein, we show that both, the N-terminal and C-terminal, regulatory domains of KEA1 reside in the chloroplast stroma and not in the intermembrane space. Considering the high homology between KEA1 and KEA2, we therefore reason that envelope KEAs must consist of an even number of transmembrane domains.

High Light Acclimation Induces Chloroplast Precursor Phosphorylation and Reduces Import Efficiency.

Acclimation is an essential process in plants on many levels, but especially in chloroplasts under changing light conditions. It is partially known how the photosynthetic machinery reacts upon exposure to high light intensities, including rearrangement of numerous protein complexes. Since the majority of proteins residing within chloroplasts needs to be posttranslationally imported into the organelles, we endeavored to study how this important process is regulated upon subjecting plants from pea and Arabidopsis to high light. Our results reveal that acclimation takes place on the one hand in the cytosol by differential phosphorylation of preproteins and resulting from the altered expression of the responsible kinases, and on the other hand at the level of the translocation machineries in the outer (TOC) and inner (TIC) envelope membranes. Intriguingly, while phosphorylation is more pronounced under high light, import itself shows a lower efficiency, along with a reduced accumulation of the Toc receptor proteins Toc34 and Toc159.

The ACT domain in chloroplast precursor-phosphorylating STY kinases binds metabolites and allosterically regulates kinase activity.

Protein import of nucleus-encoded proteins into plant chloroplasts is a highly regulated process, requiring fine-tuning mechanisms especially during chloroplast differentiation. One way of altering import efficiency is phosphorylation of chloroplast transit peptides in the cytosol. We recently investigated the role of three serine/threonine/tyrosine (STY) kinases, STY8, STY17, and STY46, in precursor phosphorylation. These three kinases have a high degree of similarity and harbor a conserved aspartate kinase-chorismate mutase-tyrA (prephenate dehydrogenase) (ACT) domain upstream of the kinase domain. The ACT domain is a widely distributed structural motif known to be important for allosteric regulation of many enzymes. In this work, using biochemical and biophysical techniques in vitro and in planta, including kinase assays, microscale thermophoresis, size exclusion chromatography, as well as site-directed mutagenesis approaches, we show that the ACT domain regulates autophosphorylation and substrate phosphorylation of the STY kinases. We found that isoleucine and S-adenosylmethionine bind to the ACT domain, negatively influencing its autophosphorylation ability. Moreover, we investigated the role of the ACT domain in planta and confirmed its involvement in chloroplast differentiation in vivo Our results provide detailed insights into the regulation of enzyme activity by ACT domains and establish that it has a role in binding amino acid ligands during chloroplast biogenesis.

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.

The Plastidic Sugar Transporter pSuT Influences Flowering and Affects Cold Responses.

Sucrose (Suc) is one of the most important types of sugars in plants, serving inter alia as a long-distance transport molecule, a carbon and energy storage compound, an osmotically active solute, and fuel for many anabolic reactions. Suc biosynthesis and degradation pathways are well known; however, the regulation of Suc intracellular distribution is poorly understood. In particular, the cellular function of chloroplast Suc reserves and the transporters involved in accumulating these substantial Suc levels remain uncharacterized. Here, we characterize the plastidic sugar transporter (pSuT) in Arabidopsis (Arabidopsis thaliana), which belongs to a subfamily of the monosaccharide transporter-like family. Transport analyses with yeast cells expressing a truncated, vacuole-targeted version of pSuT indicate that both glucose and Suc act as substrates, and nonaqueous fractionation supports a role for pSuT in Suc export from the chloroplast. The latter process is required for a correct transition from vegetative to reproductive growth and influences inflorescence architecture. Moreover, pSuT activity affects freezing-induced electrolyte release. These data further underline the central function of the chloroplast for plant development and the modulation of stress tolerance.

En route into chloroplasts: preproteins' way home.

Chloroplasts are the characteristic endosymbiotic organelles of plant cells which during the course of evolution lost most of their genetic information to the nucleus. Thus, they critically depend on the host cell for allocation of nearly their complete protein supply. This includes gene expression, translation, protein targeting, and transport-all of which need to be tightly regulated and perfectly coordinated to accommodate the cells' needs. To this end, multiple signaling pathways have been implemented that interchange information between the different cellular compartments. One of the most complex and energy consuming processes is the translocation of chloroplast-destined proteins into their target organelle. It is a concerted effort from chaperones, receptor proteins, channels, and regulatory elements to ensure correct targeting, efficient transport, and subsequent folding. Although we have discovered and learned a lot about protein import into chloroplasts in the last decades, there are still many open questions and debates about the roles of individual proteins as well as the mechanistic details. In this review, I will summarize and discuss the published data with a focus on the translocation complex in the chloroplast inner envelope membrane.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities.

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can - in contrast to fluorescent lights - be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Arabidopsis FNRL protein is an NADPH-dependent chloroplast oxidoreductase resembling bacterial ferredoxin-NADP+ reductases.

Plastidic ferredoxin-NADP+ oxidoreductases (FNRs; EC:1.18.1.2) together with bacterial type FNRs (FPRs) form the plant-type FNR family. Members of this group contain a two-domain scaffold that forms the basis of an extended superfamily of flavin adenine dinucleotide (FAD) dependent oxidoreductases. In this study, we show that the Arabidopsis thaliana At1g15140 [Ferredoxin-NADP+ oxidoreductase-like (FNRL)] is an FAD-containing NADPH dependent oxidoreductase present in the chloroplast stroma. Determination of the kinetic parameters using the DCPIP NADPH-dependent diaphorase assay revealed that the reaction catalysed by a recombinant FNRL protein followed a saturation Michaelis-Menten profile on the NADPH concentration with kcat = 3.2 ± 0.2 s-1 , KmNADPH = 1.6 ± 0.3 µM and kcat /KmNADPH = 2.0 ± 0.4 µM-1 s-1 . Biochemical assays suggested that FNRL is not likely to interact with Arabidopsis ferredoxin 1, which is supported by the sequence analysis implying that the known Fd-binding residues in plastidic FNRs differ from those of FNRL. In addition, based on structural modelling FNRL has an FAD-binding N-terminal domain built from a six-stranded ß-sheet and one α-helix, and a C-terminal NADP+ -binding α/ß domain with a five-stranded ß-sheet with a pair of α-helices on each side. The FAD-binding site is highly hydrophobic and predicted to bind FAD in a bent conformation typically seen in bacterial FPRs.

Comparative Phenotypical and Molecular Analyses of Arabidopsis Grown under Fluorescent and LED Light.

Comparative analyses of phenotypic and molecular traits of Arabidopsis thaliana grown under standardised conditions is still a challenge using climatic devices supplied with common light sources. These are in most cases fluorescent lights, which have several disadvantages such as heat production at higher light intensities, an invariable spectral output, and relatively rapid "ageing". This results in non-desired variations of growth conditions and lowers the comparability of data acquired over extended time periods. In this study, we investigated the growth behaviour of Arabidopsis Col0 under different light conditions, applying fluorescent compared to LED lamps, and we conducted physiological as well as gene expression analyses. By changing the spectral composition and/or light intensity of LEDs we can clearly influence the growth behaviour of Arabidopsis and thereby study phenotypic attributes under very specific light conditions that are stable and reproducible, which is not necessarily given for fluorescent lamps. By using LED lights, we can also roughly mimic the sun light emission spectrum, enabling us to study plant growth in a more natural-like light set-up. We observed distinct growth behaviour under the different light regimes which was reflected by physiological properties of the plants. In conclusion, LEDs provide variable emission spectra for studying plant growth under defined, stable light conditions.

Import of Soluble Proteins into Chloroplasts and Potential Regulatory Mechanisms.

Chloroplasts originated from an endosymbiotic event in which a free-living cyanobacterium was engulfed by an ancestral eukaryotic host. During evolution the majority of the chloroplast genetic information was transferred to the host cell nucleus. As a consequence, proteins formerly encoded by the chloroplast genome are now translated in the cytosol and must be subsequently imported into the chloroplast. This process involves three steps: (i) cytosolic sorting procedures, (ii) binding to the designated receptor-equipped target organelle and (iii) the consecutive translocation process. During import, proteins have to overcome the two barriers of the chloroplast envelope, namely the outer envelope membrane (OEM) and the inner envelope membrane (IEM). In the majority of cases, this is facilitated by two distinct multiprotein complexes, located in the OEM and IEM, respectively, designated TOC and TIC. Plants are constantly exposed to fluctuating environmental conditions such as temperature and light and must therefore regulate protein composition within the chloroplast to ensure optimal functioning of elementary processes such as photosynthesis. In this review we will discuss the recent models of each individual import stage with regard to short-term strategies that plants might use to potentially acclimate to changes in their environmental conditions and preserve the chloroplast protein homeostasis.

Once upon a Time - Chloroplast Protein Import Research from Infancy to Future Challenges.

Protein import into chloroplasts has been a focus of research for several decades. The first publications dealing with this fascinating topic appeared in the 1970s. From the initial realization that many plastid proteins are being encoded for in the nucleus and require transport into their target organelle to the identification of import components in the cytosol, chloroplast envelopes, and stroma, as well as elucidation of some mechanistic details, more fascinating aspects are still being unraveled. With this overview, we present a survey of the beginnings of chloroplast protein import research, the first steps on this winding road, and end with a glimpse into the future.

The extreme Albino3 (Alb3) C terminus is required for Alb3 stability and function in Arabidopsis thaliana.

MAIN CONCLUSION: The extreme Alb3 C terminus is important for Alb3 stability in a light dependent manner, but is dispensable for LHCP insertion or D1 synthesis. YidC/Oxa1/Alb3 dependent insertion of membrane proteins is evolutionary conserved among bacteria, mitochondria and chloroplasts. Chloroplasts are challenged by the need to coordinate membrane integration of nuclear encoded, post-translationally targeted proteins into the thylakoids as well as of proteins translated on plastid ribosomes. The pathway facilitating post-translational targeting of the light-harvesting chlorophyll a/b binding proteins involves the chloroplast signal recognition particle, cpSRP54 and cpSRP43, as well as its membrane receptor FtsY and the translocase Alb3. Interaction of cpSRP43 with Alb3 is mediated by the positively charged, stromal exposed C terminus of Alb3. In this study, we utilized an Alb3 T-DNA insertion mutant in Arabidopsis thaliana lacking the last 75 amino acids to elucidate the function of this domain (alb3∆C). However, the truncated Alb3 protein (Alb3∆C) proved to be unstable under standard growth conditions, resulting in a reduction of Alb3∆C to 20 % of wild-type levels. In contrast, accumulation of Alb3∆C was comparable to wild type under low light growth conditions. Alb3∆C mutants grown under low light conditions were only slightly paler than wild type, accumulated almost wild-type levels of light harvesting proteins and were not affected in D1 synthesis, therefore showing that the extreme Alb3 C terminus is dispensable for both, co- and post-translational, protein insertion into the thylakoid membrane. However, reduction of Alb3∆C levels as observed under standard growth conditions resulted not only in a severely diminished accumulation of all thylakoid complexes but also in a strong defect in D1 synthesis and membrane insertion.