An RNA thermometer in the chloroplast genome of Chlamydomonas facilitates temperature-controlled gene expression.

Riboregulators such as riboswitches and RNA thermometers provide simple, protein-independent tools to control gene expression at the post-transcriptional level. In bacteria, RNA thermometers regulate protein synthesis in response to temperature shifts. Thermometers outside of the bacterial world are rare, and in organellar genomes, no RNA thermometers have been identified to date. Here we report the discovery of an RNA thermometer in a chloroplast gene of the unicellular green alga Chlamydomonas reinhardtii. The thermometer, residing in the 5' untranslated region of the psaA messenger RNA forms a hairpin-type secondary structure that masks the Shine-Dalgarno sequence at 25°C. At 40°C, melting of the secondary structure increases accessibility of the Shine-Dalgarno sequence to initiating ribosomes, thus enhancing protein synthesis. By targeted nucleotide substitutions and transfer of the thermometer into Escherichia coli, we show that the secondary structure is necessary and sufficient to confer the thermometer properties. We also demonstrate that the thermometer provides a valuable tool for inducible transgene expression from the Chlamydomonas plastid genome, in that a simple temperature shift of the algal culture can greatly increase recombinant protein yields.

Arabidopsis AUTOPHAGY-RELATED2 is essential for ATG18a and ATG9 trafficking during autophagosome closure.

As a fundamental metabolic pathway, autophagy plays important roles in plant growth and development, particularly under stress conditions. A set of autophagy-related (ATG) proteins is recruited for the formation of a double-membrane autophagosome. Among them, the essential roles of ATG2, ATG18, and ATG9 have been well established in plant autophagy via genetic analysis; however, the underlying molecular mechanism for ATG2 in plant autophagosome formation remains poorly understood. In this study, we focused on the specific role of ATG2 in the trafficking of ATG18a and ATG9 during autophagy in Arabidopsis (Arabidopsis thaliana). Under normal conditions, YFP-ATG18a proteins are partially localized on late endosomes and translocated to ATG8e-labeled autophagosomes upon autophagic induction. Real-time imaging analysis revealed sequential recruitment of ATG18a on the phagophore membrane, showing that ATG18a specifically decorated the closing edges and finally disassociated from the completed autophagosome. However, in the absence of ATG2, most of the YFP-ATG18a proteins are arrested on autophagosomal membranes. Ultrastructural and 3D tomography analysis showed that unclosed autophagosome structures are accumulated in the atg2 mutant, displaying direct connections with the endoplasmic reticulum membrane and vesicular structures. Dynamic analysis of ATG9 vesicles suggested that ATG2 depletion also affects the association between ATG9 vesicles and the autophagosomal membrane. Furthermore, using interaction and recruitment analysis, we mapped the interaction relationship between ATG2 and ATG18a, implying a possible role of ATG18a in recruiting ATG2 and ATG9 to the membrane. Our findings unveil a specific role of ATG2 in coordinating ATG18a and ATG9 trafficking to mediate autophagosome closure in Arabidopsis.

Signal motif-dependent ER export of the Qc-SNARE BET12 interacts with MEMB12 and affects PR1 trafficking in Arabidopsis.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are well-known for their role in controlling membrane fusion, the final, but crucial step, in vesicular transport in eukaryotes. SNARE proteins contribute to various biological processes including pathogen defense and channel activity regulation, as well as plant growth and development. Precise targeting of SNARE proteins to destined compartments is a prerequisite for their proper functioning. However, the underlying mechanism(s) for SNARE targeting in plants remains obscure. Here, we investigate the targeting mechanism of the Arabidopsis thaliana Qc-SNARE BET12, which is involved in protein trafficking in the early secretory pathway. Two distinct signal motifs that are required for efficient BET12 ER export were identified. Pulldown assays and in vivo imaging implicated that both the COPI and COPII pathways were required for BET12 targeting. Further studies using an ER-export-defective form of BET12 revealed that the Golgi-localized Qb-SNARE MEMB12, a negative regulator of pathogenesis-related protein 1 (PR1; At2g14610) secretion, was its interacting partner. Ectopic expression of BET12 caused no inhibition in the general ER-Golgi anterograde transport but caused intracellular accumulation of PR1, suggesting that BET12 has a regulatory role in PR1 trafficking in A. thaliana.

ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis.

Autophagy is a conserved pathway for bulk degradation of cytoplasmic material by a double-membrane structure named the autophagosome. The initiation of autophagosome formation requires the recruitment of autophagy-related protein 9 (ATG9) vesicles to the preautophagosomal structure. However, the functional relationship between ATG9 vesicles and the phagophore is controversial in different systems, and the molecular function of ATG9 remains unknown in plants. Here, we demonstrate that ATG9 is essential for endoplasmic reticulum (ER)-derived autophagosome formation in plants. Through a combination of genetic, in vivo imaging and electron tomography approaches, we show that Arabidopsis ATG9 deficiency leads to a drastic accumulation of autophagosome-related tubular structures in direct membrane continuity with the ER upon autophagic induction. Dynamic analyses demonstrate a transient membrane association between ATG9 vesicles and the autophagosomal membrane during autophagy. Furthermore, trafficking of ATG18a is compromised in atg9 mutants during autophagy by forming extended tubules in a phosphatidylinositol 3-phosphate-dependent manner. Taken together, this study provides evidence for a pivotal role of ATG9 in regulating autophagosome progression from the ER membrane in Arabidopsis.

Unique COPII component AtSar1a/AtSec23a pair is required for the distinct function of protein ER export in Arabidopsis thaliana.

Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on α-amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes.

Protein secretion in plants: conventional and unconventional pathways and new techniques.

Protein secretion is an essential process in all eukaryotic cells and its mechanisms have been extensively studied. Proteins with an N-terminal leading sequence or transmembrane domain are delivered through the conventional protein secretion (CPS) pathway from the endoplasmic reticulum (ER) to the Golgi apparatus. This feature is conserved in yeast, animals, and plants. In contrast, the transport of leaderless secretory proteins (LSPs) from the cytosol to the cell exterior is accomplished via the unconventional protein secretion (UPS) pathway. So far, the CPS pathway has been well characterized in plants, with several recent studies providing new information about the regulatory mechanisms involved. On the other hand, studies on UPS pathways in plants remain descriptive, although a connection between UPS and the plant defense response is becoming more and more apparent. In this review, we present an update on CPS and UPS. With the emergence of new techniques, a more comprehensive understanding of protein secretion in plants can be expected in the future.

Identification and characterization of the COPII vesicle-forming GTPase Sar1 in Chlamydomonas.

Eukaryotic cells are highly compartmentalized, requiring elaborate transport mechanisms to facilitate the movement of proteins between membrane-bound compartments. Most proteins synthesized in the endoplasmic reticulum (ER) are transported to the Golgi apparatus through COPII-mediated vesicular trafficking. Sar1, a small GTPase that facilitates the formation of COPII vesicles, plays a critical role in the early steps of this protein secretory pathway. Sar1 was characterized in yeast, animals and plants, but no Sar1 homolog has been identified and functionally analyzed in algae. Here we identified a putative Sar1 homolog (CrSar1) in the model green alga Chlamydomonas reinhardtii through amino acid sequence similarity. We employed site-directed mutagenesis to generate a dominant-negative mutant of CrSar1 (CrSar1DN). Using protein secretion assays, we demonstrate the inhibitory effect of CrSar1DN on protein secretion. However, different from previously studied organisms, ectopic expression of CrSar1DN did not result in collapse of the ER-Golgi interface in Chlamydomonas. Nonetheless, our data suggest a largely conserved role of CrSar1 in the ER-to-Golgi protein secretory pathway in green algae.

Control of plastid inheritance by environmental and genetic factors.

The genomes of cytoplasmic organelles (mitochondria and plastids) are maternally inherited in most eukaryotes, thus excluding organellar genomes from the benefits of sexual reproduction and recombination. The mechanisms underlying maternal inheritance are largely unknown. Here we demonstrate that two independently acting mechanisms ensure maternal inheritance of the plastid (chloroplast) genome. Conducting large-scale genetic screens for paternal plastid transmission, we discovered that mild chilling stress during male gametogenesis leads to increased entry of paternal plastids into sperm cells and strongly increased paternal plastid transmission. We further show that the inheritance of paternal plastid genomes is controlled by the activity of a genome-degrading exonuclease during pollen maturation. Our data reveal that (1) maternal inheritance breaks down under specific environmental conditions, (2) an organelle exclusion mechanism and a genome degradation mechanism act in concert to prevent paternal transmission of plastid genes and (3) plastid inheritance is determined by complex gene-environment interactions.

Mechanistic insights into an atypical interaction between ATG8 and SH3P2 in Arabidopsis thaliana.

In selective macroautophagy/autophagy, cargo receptors are recruited to the forming autophagosome by interacting with Atg8 (autophagy-related 8)-family proteins and facilitate the selective sequestration of specific cargoes for autophagic degradation. In addition, Atg8 interacts with a number of adaptors essential for autophagosome biogenesis, including ATG and non-ATG proteins. The majority of these adaptors and receptors are characterized by an Atg8-family interacting motif (AIM) for binding to Atg8. However, the molecular basis for the interaction mode between ATG8 and regulators or cargo receptors in plants remains largely unclear. In this study, we unveiled an atypical interaction mode for Arabidopsis ATG8f with a plant unique adaptor protein, SH3P2 (SH3 domain-containing protein 2), but not with the other two SH3 proteins. By structure analysis of the unbound form of ATG8f, we identified the unique conformational changes in ATG8f upon binding to the AIM sequence of a plant known autophagic receptor, NBR1. To compare the binding affinity of SH3P2-ATG8f with that of ATG8f-NBR1, we performed a gel filtration assay to show that ubiquitin-associated domain of NBR1 outcompetes the SH3 domain of SH3P2 for ATG8f interaction. Biochemical and cellular analysis revealed that distinct interfaces were employed by ATG8f to interact with NBR1 and SH3P2. Further subcellular analysis showed that the AIM-like motif of SH3P2 is essential for its recruitment to the phagophore membrane but is dispensable for its trafficking in endocytosis. Taken together, our study provides an insightful structural basis for the ATG8 binding specificity toward a plant-specific autophagic adaptor and a conserved autophagic receptor.Abbreviations: ATG, autophagy-related; AIM, Atg8-family interacting motif; BAR, Bin-Amphiphysin-Rvs; BFA, brefeldin A; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; CCV, clathrin-coated-vesicle; CLC2, clathrin light chain 2; Conc A, concanamycin A; ER, endoplasmic reticulum; LDS, LIR docking site; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; PE, phosphatidylethanolamine; SH3P2, SH3 domain containing protein 2; SH3, Src-Homology-3; UBA, ubiquitin-associated; UIM, ubiquitin-interacting motif.

Autophagosome Biogenesis and the Endoplasmic Reticulum: A Plant Perspective.

The autophagosome is a double-membrane compartment formed during autophagy that sequesters and delivers cargoes for their degradation or recycling into the vacuole. Analyses of the AuTophaGy-related (ATG) proteins have unveiled dynamic mechanisms for autophagosome biogenesis. Recent advances in plant autophagy research highlight a complex interplay between autophagosome biogenesis and the endoplasmic reticulum (ER): on the one hand ER serves as a membrane source for autophagosome initiation and a signaling platform for autophagy regulation; on the other hand ER turnover is connected to selective autophagy. We provide here an integrated view of ER-based autophagosome biogenesis in plants in comparison with the newest findings in yeast and mammals, with an emphasis on the hierarchy of the core ATG proteins, ATG9 trafficking, and ER-resident regulators in autophagy.

An Overview of Protein Secretion in Plant Cells.

The delivery of proteins to the apoplast or protein secretion is an essential process in plant cells. Proteins are secreted to perform various biological functions such as cell wall modification and defense response. Conserved from yeast to mammals, both conventional and unconventional protein secretion pathways have been demonstrated in plants. In the conventional protein secretion pathway, secretory proteins with an N-terminal signal peptide are transported to the extracellular region via the endoplasmic reticulum-Golgi apparatus and the subsequent endomembrane system. By contrast, multiple unconventional protein secretion pathways are proposed to mediate the secretion of the leaderless secretory proteins. In this review, we summarize the recent findings and provide a comprehensive overview of protein secretion pathways in plant cells.

COPII Paralogs in Plants: Functional Redundancy or Diversity?

In eukaryotes, the best-described mechanism of endoplasmic reticulum (ER) export is mediated by coat protein complex II (COPII) vesicles, which comprise five conserved cytosolic components [secretion-associated, Ras-related protein 1 (Sar1), Sec23-24, and Sec13-31]. In higher organisms, multiple paralogs of COPII components are created due to gene duplication. However, the functional diversity of plant COPII subunit isoforms remains largely elusive. Here we summarize and discuss the latest findings derived from studies of various arabidopsis COPII subunit isoforms and their functional diversity. We also put forward testable hypotheses on distinct populations of COPII vesicles performing unique functions in ER export in developmental and stress-related pathways in plants.

Origin of the Autophagosomal Membrane in Plants.

During autophagy, cargo molecules destined for degradation are sequestrated into a double-membrane structure called autophagosome, which subsequently fuses with the vacuole. An isolation membrane structure (also called the phagophore) initiates from the platform termed PAS (phagophore assembly site or preautophagosomal structure), which then elongates and expands to become the completed autophagosome. The origin of the membrane for autophagosome formation has been extensively investigated but remains an enigma in the field of autophagy. In yeast and mammalian cells multiple membrane sources have been suggested to contribute to autophagosome formation at different steps, from initiation through expansion and maturation. Recent studies in plants have provided a significant advance in our understanding of the conserved role of autophagy and the underlying mechanism for autophagosome formation. Here, we will discuss and evaluate these new findings on autophagosome formation in plants, with a particular focus on the origin of plant autophagosomal membranes.