Chloroplast Targeting Research Articles

The N-region sequence context impacts the chloroplast import efficiency of multi-TMD protein.

Targeting heterologous multi-transmembrane domain (TMD) proteins to plant chloroplasts requires sequences in addition to the chloroplast transit peptide (cTP). The N-terminal domain (N-region), located C-terminal to the cTP in chloroplast inner envelope membrane proteins, is an essential region for import. However, it was unclear if the N-region functions solely as a spacer sequence to facilitate cTP access or if it plays an active role in the import process. This study addresses the N-region's role by using combinations of cTPs and N-regions from Arabidopsis chloroplast inner envelope membrane proteins to direct the cyanobacterial protein SbtA to the chloroplast. We find that the sequence context of the N-region affects the chloroplast import efficiency of SbtA, with particular sequences mis-targeting the protein to different cellular sub-compartments. Additionally, specific cTP and N-region pairs exhibit varying targeting efficiencies for different heterologous proteins. Substituting individual N-region motifs did not significantly alter the chloroplast targeting efficiency of a particular cTP and N-region pair. We conclude that the N-region exhibits contextual functioning and potentially functional redundancy in motifs.

Identification and preliminary characterization of conserved uncharacterized proteins from Chlamydomonas reinhardtii, Arabidopsis thaliana, and Setaria viridis.

The rapid accumulation of sequenced plant genomes in the past decade has outpaced the still difficult problem of genome-wide protein-coding gene annotation. A substantial fraction of protein-coding genes in all plant genomes are poorly annotated or unannotated and remain functionally uncharacterized. We identified unannotated proteins in three model organisms representing distinct branches of the green lineage (Viridiplantae): Arabidopsis thaliana (eudicot), Setaria viridis (monocot), and Chlamydomonas reinhardtii (Chlorophyte alga). Using similarity searching, we identified a subset of unannotated proteins that were conserved between these species and defined them as Deep Green proteins. Bioinformatic, genomic, and structural predictions were performed to begin classifying Deep Green genes and proteins. Compared to whole proteomes for each species, the Deep Green set was enriched for proteins with predicted chloroplast targeting signals predictive of photosynthetic or plastid functions, a result that was consistent with enrichment for daylight phase diurnal expression patterning. Structural predictions using AlphaFold and comparisons to known structures showed that a significant proportion of Deep Green proteins may possess novel folds. Though only available for three organisms, the Deep Green genes and proteins provide a starting resource of high-value targets for further investigation of potentially new protein structures and functions conserved across the green lineage.

Engineering Nannochloropsis oceanica for the production of diterpenoid compounds.

Photosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable, light-driven biofactories for the production of high-value natural products such as terpenoids. Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how N. oceanica can be engineered to produce the diterpene casbene-an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from Daphne genkwa (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g-1 total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate-limiting enzymes in the 2-C-methyl- d-erythritol 4-phosphatepathway, Coleus forskohlii 1-deoxy- d-xylulose-5-phosphate synthaseand geranylgeranyl diphosphate synthase genes,further enhanced the yield of casbeneto a titer up to 1.80 mg g-1 DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.

Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D

The polyextremophilic Cyanidiophyceae are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal β-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments.

The journey of preproteins across the chloroplast membrane systems.

The photosynthetic capacity of chloroplasts is vital for autotrophic growth in algae and plants. The origin of the chloroplast has been explained by the endosymbiotic theory that proposes the engulfment of a cyanobacterium by an ancestral eukaryotic cell followed by the transfer of many cyanobacterial genes to the host nucleus. As a result of the gene transfer, the now nuclear-encoded proteins acquired chloroplast targeting peptides (known as transit peptides; transit peptide) and are translated as preproteins in the cytosol. Transit peptides contain specific motifs and domains initially recognized by cytosolic factors followed by the chloroplast import components at the outer and inner envelope of the chloroplast membrane. Once the preprotein emerges on the stromal side of the chloroplast protein import machinery, the transit peptide is cleaved by stromal processing peptidase. In the case of thylakoid-localized proteins, cleavage of the transit peptides may expose a second targeting signal guiding the protein to the thylakoid lumen or allow insertion into the thylakoid membrane by internal sequence information. This review summarizes the common features of targeting sequences and describes their role in routing preproteins to and across the chloroplast envelope as well as the thylakoid membrane and lumen.

Phenoloxidases: catechol oxidase - the temporary employer and laccase - the rising star of vascular plants.

Phenolics are vital for the adaptation of plants to terrestrial habitats and for species diversity. Phenoloxidases (catechol oxidases, COs, and laccases, LACs) are responsible for the oxidation and polymerization of phenolics. However, their origin, evolution, and differential roles during plant development and land colonization are unclear. We performed the phylogeny, domain, amino acids, compositional biases, and intron analyses to clarify the origin and evolution of COs and LACs, and analysed the structure, selective pressure, and chloroplast targeting to understand the species-dependent distribution of COs. We found that Streptophyta COs were not homologous to the Chlorophyta tyrosinases (TYRs), and might have been acquired by horizontal gene transfer from bacteria. COs expanded in bryophytes. Structural-functionality and selective pressure were partially responsible for the species-dependent retention of COs in embryophytes. LACs emerged in Zygnemaphyceae, having evolved from ascorbate oxidases (AAOs), and prevailed in the vascular plants and strongly expanded in seed plants. COs and LACs coevolved with the phenolic metabolism pathway genes. These results suggested that TYRs and AAOs were the first-stage phenoloxidases in Chlorophyta. COs might be the second key for the early land colonization. LACs were the third one (dominating in the vascular plants) and might be advantageous for diversified phenol substrates and the erect growth of plants. This work provided new insights into how phenoloxidases evolved and were devoted to plant evolution.

Translation rate underpins specific targeting of N-terminal transmembrane proteins to mitochondria.

Protein biogenesis is a complex process, and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles. To direct targeting, organellar proteins carry an organelle-specific targeting signal for recognition by organelle-specific import machinery. However, the situation is confusing for transmembrane domain (TMD)-containing signal anchored (SA) proteins of various organelles because TMDs function as an endoplasmic reticulum (ER) targeting signal. Although ER targeting of SA proteins is well understood, how they are targeted to mitochondria and chloroplasts remains elusive. Here, we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts. Mitochondrial targeting requires multiple motifs around and within TMDs; a basic residue and an arginine-rich region flanking the N- and C-termini of TMDs, respectively, and an aromatic residue in the C-terminal side of the TMD that specify mitochondrial targeting in an additive manner. These motifs play a role in slowing down the elongation speed during translation, thereby ensuring mitochondrial targeting in a co-translational manner. By contrast, the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a posttranslational manner. This article is protected by copyright. All rights reserved.

Converting antimicrobial into targeting peptides reveals key features governing protein import into mitochondria and chloroplasts

We asked what peptide features govern targeting to the mitochondria versus the chloroplast, using antimicrobial peptides as a starting point. This approach was inspired by the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell, to which organelle progenitors became resistant. To explore the molecular changes required to convert antimicrobial into targeting peptides, we expressed a set of 13 antimicrobial peptides in Chlamydomonas reinhardtii. Peptides were systematically modified to test distinctive features of mitochondrion- and chloroplast-targeting peptides, and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as a cargo protein. Mitochondrial targeting can be achieved by some unmodified antimicrobial peptide sequences. Targeting to both organelles is improved by replacing lysines with arginines. Chloroplast targeting is enabled by the presence of flanking unstructured sequences, additional constraints consistent with chloroplast endosymbiosis having occurred in a cell that already contained mitochondria. If indeed targeting peptides evolved from antimicrobial peptides, then required modifications imply a temporal evolutionary scenario with an early exchange of cationic residues and a late acquisition of chloroplast-specific motifs.

Magnaporthe oryzae Chloroplast Targeting Endo-β-1,4-Xylanase I MoXYL1A Regulates Conidiation, Appressorium Maturation and Virulence of the Rice Blast Fungus

Endo-β-1,4-Xylanases are a group of extracellular enzymes that catalyze the hydrolysis of xylan, a principal constituent of the plant primary cell wall. The contribution of Endo-β-1,4-Xylanase I to both physiology and pathogenesis of the rice blast fungus M. oryzae is unknown. Here, we characterized the biological function of two endoxylanase I (MoXYL1A and MoXYL1B) genes in the development of M. oryzae using targeted gene deletion, biochemical analysis, and fluorescence microscopy. Phenotypic analysis of ∆Moxyl1A strains showed that MoXYL1A is required for the full virulence of M. oryzae but is dispensable for the vegetative growth of the rice blast fungus. MoXYL1B, in contrast, did not have a clear role in the infectious cycle but has a critical function in asexual reproduction of the fungus. The double deletion mutant was severely impaired in pathogenicity and virulence as well as asexual development. We found that MoXYL1A deletion compromised appressorium morphogenesis and function, leading to failure to penetrate host cells. Fluorescently tagged MoXYL1A and MoXYL1B displayed cytoplasmic localization in M. oryzae, while analysis of MoXYL1A-GFP and MoXYL1B-GFP in-planta revealed translocation and accumulation of these effector proteins into host cells. Meanwhile, sequence feature analysis showed that MoXYL1A possesses a transient chloroplast targeting signal peptide, and results from an Agrobacterium infiltration assay confirmed co-localization of MoXYL1A-GFP with ChCPN10C-RFP in the chloroplasts of host cells. MoXYL1B, accumulated to the cytoplasm of the host. Taken together, we conclude that MoXYL1A is a secreted effector protein that likely promotes the virulence of M. oryzae by interfering in the proper functioning of the host chloroplast, while the related xylanase MoXYL1B does not have a major role in virulence of M. oryzae.

Expression of nuclear-encoded, haptophyte-derived ftsH genes support extremely rapid PSII repair and high-light photoacclimation in Karenia brevis (Dinophyceae)

Karenia brevis, a neurotoxic dinoflagellate that produces brevetoxins, is endemic to the Gulf of Mexico and can grow at high irradiances typical of surface waters found there. To build upon a growing number of studies addressing high-light tolerance in K. brevis, specific photobiology and molecular mechanisms underlying this capacity were evaluated in culture. Since photosystem II (PSII) repair cycle activity can be crucial to high light tolerance in plants and algae, the present study assessed this capacity in K. brevis and characterized the ftsH-like genes which are fundamental to this process. Compared with cultures grown in low-light, cultures grown in high-light showed a 65-fold increase in PSII photoinactivation, a ∼50-fold increase in PSII repair, enhanced nonphotochemical quenching (NPQ), and depressed Fv/Fm. Repair rates were among the fastest reported in phytoplankton. Publicly available K. brevis transcriptomes (MMETSP) were queried for ftsH-like sequences and refined with additional sequencing from two K. brevis strains. The genes were phylogenetically related to haptophyte orthologs, implicating acquisition during tertiary endosymbiosis. RT-qPCR of three of the four ftsH-like homologs revealed that poly-A tails predominated in all homologs, and that the most highly expressed homolog had a 5′ splice leader and amino-acid motifs characteristic of chloroplast targeting, indicating nuclear encoding for this plastid-targeted gene. High-light cultures showed a ∼1.5-fold upregulation in mRNA expression of the thylakoid-associated genes. Overall, in conjunction with NPQ mechanisms, rapid PSII repair mediated by a haptophyte-derived ftsH prevents chronic photoinhibition in K. brevis. Our findings continue to build the case that high-light photobiology—supported by the acquisition and maintenance of tertiary endosymbiotic genes—is critical to the success of K. brevis in the Gulf of Mexico.

Both metaxin and Tom20 together with two mitochondria-specific motifs support mitochondrial targeting of dual-targeting AtSufE1.

Plant cells have two endosymbiotic organelles, chloroplasts, and mitochondria. These organelles perform specific functions that depend on organelle-specific proteins. The majority of chloroplast and mitochondrial proteins are specifically imported by the transit peptide and presequence, respectively. However, a significant number of proteins are also dually targeted to these two organelles. Currently, it is not fully understood how proteins are dually targeted to both chloroplasts and mitochondria. In this study, the mechanism underlying mitochondrial targeting of dual targeting AtSufE1 in Arabidopsis was elucidated. The N-terminal fragment containing 80 residues of AtSufE1 (AtSufE1N80) was sufficient to confer dual targeting of reporter protein, AtSufE1N80:GFP, in protoplasts. Two sequence motifs, two arginine residues at 15th and 21st positions, and amino acid (aa) sequence motif AKTLLLRPLK from the 31st to 40th aa position, were responsible for targeting to mitochondria a portion of reporter proteins amid the chloroplast targeting. The sequence motif PSEVPFRRT from the 41st to 50th aa position constitutes a common motif for targeting to both chloroplasts and mitochondria. For mitochondrial import of AtSufE1:N80, Metaxin played a critical role. In addition, BiFC and protein pull-down experiments showed that AtSufE1N80 specifically interacts with import receptors, Metaxin and Tom20. The interaction of AtSufE1N80 with Metaxin was required for the interaction with Tom20. Based on these results, we propose that mitochondrial targeting of dual-targeting AtSufE1 is mediated by both mitochondria-specific and common sequence motifs in the signal sequence through the interaction with import receptors, Metaxin and Tom20, in a successive manner.

Cell-penetrating peptide for targeted macromolecule delivery into plant chloroplasts.

Reports on chloroplast-targeted protein delivery using cell-penetrating peptides are scarce. In this study, a novel peptide-based macromolecule delivery strategy targeting chloroplasts was successfully developed in wheat mesophyll protoplasts. A peptide derived from the signal sequence of the chloroplast-targeted protein of ferredoxin-thioredoxin reductase catalytic chain of Spinacia oleracea with UniProtKB Id-P41348 exhibits properties of cellular internalization. DNase I was efficiently delivered into the chloroplast using 10 μM cTP with an efficiency of more than 90%. This cell-penetrating peptide-mediated approach offers various advantages over the existing chloroplast targeting methods, such as non-invasiveness, biocompatibility, low-toxicity, and target-specific delivery. The present study shows that peptide-based strategies hold tremendous potential in the field of chloroplast biotechnology. KEY POINTS: • Screening of database of chloroplast targeting peptides in order to develop an efficient cell-penetrating peptide termed as cTP. • cTP efficiently crosses the cell barrier and demonstrated chloroplast-localization. • cTP can be incorporated as a promising strategy for delivering macromolecules for crop improvement.

Liquid-Liquid Phase Separation Phenomenon on Protein Sorting Within Chloroplasts.

In higher plants, chloroplasts are vital organelles possessing highly complex compartmentalization. As most chloroplast-located proteins are encoded in the nucleus and synthesized in the cytosol, the correct sorting of these proteins to appropriate compartments is critical for the proper functions of chloroplasts as well as plant survival. Nuclear-encoded chloroplast proteins are imported into stroma and further sorted to distinct compartments via different pathways. The proteins predicted to be sorted to the thylakoid lumen by the chloroplast twin arginine transport (cpTAT) pathway are shown to be facilitated by STT1/2 driven liquid-liquid phase separation (LLPS). Liquid-liquid phase separation is a novel mechanism to facilitate the formation of membrane-less sub-cellular compartments and accelerate biochemical reactions temporally and spatially. In this review, we introduce the sorting mechanisms within chloroplasts, and briefly summarize the properties and significance of LLPS, with an emphasis on the novel function of LLPS in the sorting of cpTAT substrate proteins. We conclude with perspectives for the future research on chloroplast protein sorting and targeting mechanisms.

The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism.

Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.

Evaluation of CBSX Proteins as Regulators of the Chloroplast Thioredoxin System.

The chloroplast-localized cystathionine β-synthase X (CBSX) proteins CBSX1 and CBSX2 have been proposed as modulators of thioredoxins (Trxs). In this study, the contribution of CBSX proteins to the redox regulation of thiol enzymes in the chloroplast Trx system was evaluated both in vitro and in vivo. The in vitro biochemical studies evaluated whether CBSX proteins alter the specificities of classical chloroplastic Trx f and Trx m for their target proteins. However, addition of CBSX proteins did not alter the specificities of Trx f and Trx m for disulfide bond reduction of the photosynthesis-related major thiol enzymes, FBPase, SBPase, and NADP-MDH. In vivo analysis showed that CBSX-deficient mutants grew similarly to wild type plants under continuous normal light conditions and that CBSX deficiency did not affect photo-reduction of photosynthesis-related thiol enzymes by Trx system at several light intensities. Although CBSX proteins have been suggested as modulators in the chloroplast Trx system, our results did not support this model, at least in the cases of FBPase, SBPase, and NADP-MDH in leaves. However, fresh weights of the cbsx2 mutants were decreased under short day. Since Trxs regulate many proteins participating in various metabolic reactions in the chloroplast, CBSX proteins may function to regulate other chloroplast Trx target proteins, or serve as modulators in non-photosynthetic plastids of flowers. As a next stage, further investigations are required to understand the modulation of Trx-dependent redox regulation by plastidal CBSX proteins.

Identification, Gene Structure, and Expression of BnMicEmUP: A Gene Upregulated in Embryogenic Brassica napus Microspores.

Microspores of Brassica napus can be diverted from normal pollen development into embryogenesis by treating them with a mild heat shock. As microspore embryogenesis closely resembles zygotic embryogenesis, it is used as model for studying the molecular mechanisms controlling embryo formation. A previous study comparing the transcriptomes of three-day-old sorted embryogenic and pollen-like (non-embryogenic) microspores identified a gene homologous to AT1G74730 of unknown function that was upregulated 8-fold in the embryogenic cells. In the current study, the gene was isolated and sequenced from B. napus and named BnMicEmUP (B. napus microspore embryogenesis upregulated gene). Four forms of BnMicEmUP mRNA and three forms of genomic DNA were identified. BnMicEmUP2,3 was upregulated more than 7-fold by day 3 in embryogenic microspore cultures compared to non-induced cultures. BnMicEmUP1,4 was highly expressed in leaves. Transient expression studies of BnMicEmUP3::GFP fusion protein in Nicotiana benthamiana and in stable Arabidopsis transgenics showed that it accumulates in chloroplasts. The features of the BnMicEmUP protein, which include a chloroplast targeting region, a basic region, and a large region containing 11 complete leucine-rich repeats, suggest that it is similar to a bZIP PEND (plastid envelope DNA-binding protein) protein, a DNA binding protein found in the inner envelope membrane of developing chloroplasts. Here, we report that the BnMicEmUP3 overexpression in Arabidopsis increases the sensitivity of seedlings to exogenous abscisic acid (ABA). The BnMicEmUP proteins appear to be transcription factors that are localized in plastids and are involved in plant responses to biotic and abiotic environmental stresses; as well as the results obtained from this study can be used to improve crop yield.

Identification and expression analysis of chloroplast ribonucleoproteins (cpRNPs) in Arabidopsis and rice.

Chloroplast ribonucleoproteins (cpRNPs) are implicated in splicing, editing, and stability control of chloroplast RNAs as well as in regulating development and stress tolerance. To facilitate a comprehensive understanding of their functions, we carried out a genome-wide identification, curation, and phylogenetic analysis of cpRNP genes in Oryza sativa (rice) and Arabidopsis thaliana (Arabidopsis). Ten cpRNP genes were identified in each of Arabidopsis and rice genomes based on the presence of two RRM (RNA-recognition motif) domains and an N-terminal chloroplast targeting signal peptide in the predicted proteins. These proteins are localized to chloroplasts. Gene expression analysis revealed that cpRNP genes have differential tissue expression patterns and some cpRNP genes are induced by abiotic stresses such as cold, heat, and drought. Taken together, our study provides a comprehensive annotation of the cpRNP gene family and their expression patterns in Arabidopsis and rice which will facilitate further studies on their roles in plant growth and stress responses.

Characterization of PtAOS1 Promoter and Three Novel Interacting Proteins Responding to Drought in Poncirus trifoliata

Jasmonic acid (JA) plays a crucial role in various biological processes including development, signal transduction and stress response. Allene oxide synthase (AOS) catalyzing (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT) to an unstable allene oxide is involved in the first step of JA biosynthesis. Here, we isolated the PtAOS1 gene and its promoter from trifoliate orange (Poncirus trifoliata). PtAOS1 contains a putative chloroplast targeting sequence in N-terminal and shows relative to pistachio (Pistacia vera) AOS. A number of stress-, light- and hormone-related cis-elements were found in the PtAOS1 promoter which may be responsible for the up-regulation of PtAOS1 under drought and JA treatments. Transient expression in tobacco (Nicotiana benthamiana) demonstrated that the P−532 (−532 to +1) fragment conferring drive activity was a core region in the PtAOS1 promoter. Using yeast one-hybrid, three novel proteins, PtDUF886, PtDUF1685 and PtRAP2.4, binding to P−532 were identified. The dual luciferase assay in tobacco illustrated that all three transcription factors could enhance PtAOS1 promoter activity. Genes PtDUF1685 and PtRAP2.4 shared an expression pattern which was induced significantly by drought stress. These findings should be available evidence for trifoliate orange responding to drought through JA modulation.

Ribosome rescue activity of an Arabidopsis thaliana ArfB homolog.

A homolog of the bacterial ribosome rescue factor ArfB was identified in Arabidopsis thaliana. The factor, named AtArfB for Arabidopsis thaliana ArfB, showed ribosome rescue activity in both in vivo and in vitro assays based on the bacterial translation system. As has been shown for ArfB, the ribosome rescue activity of AtArfB was dependent on the GGQ motif, the crucial motif for the function of class I release factors and ArfB. The C-terminal region of AtArfB was also important for its function. The N-terminal region of AtArfB, which is absent in bacterial ArfB, functioned as a transit peptide for chloroplast targeting in tobacco cells. These results strongly suggest that AtArfB is a ribosome rescue factor that functions in chloroplasts.

The Function and Regulation of Chloroplast Targeting of AZI1, a Key Factor in Plant Systemic Immune Signaling

Recognition of microbes and microbe‐associated molecular patterns by plant cell receptors induces signaling cascades that prime broad‐spectrum defense responses to suppress subsequent infections. Primed plants can activate stronger defense responses more quickly than unprimed plants when challenged by pathogens and other stresses. Systemic priming requires the long‐distance movement of signals produced during initial exposure events. Distinct responses are induced depending on whether roots are colonized by specific commensal microbes or if leaves are infected by certain pathogens. The protein AZI1, a member of the lipid transfer protein superfamily, is essential for both root‐stimulated and leaf‐stimulated defense priming. AZI1 is also needed for uptake and mobilization of azelaic acid (AZA). A defense signal derived from chloroplast lipid oxidation, the oxylipin AZA stimulates priming when applied to leaves or roots.Functional AZI1‐GFP traffics among chloroplast outer envelope membranes (OEMs), ER, PM, and intercellular channels called plasmodesmata. The targeting of AZI1 to chloroplast OEMs requires a newly described bipartite protein targeting signal featuring an N‐terminal hydrophobic domain and an internal region called the proline‐rich region. AZI1’s association with chloroplasts and plasmodesmata is suggestive of a pathway by which AZI1 may act in the intercellular mobilization of a chloroplast‐derived lipid signal(s) such as AZA. Consistent with this hypothesis, AZI1 increasingly targets to chloroplasts under infection conditions. This infection‐induced chloroplast accumulation requires the kinase MPK3, which, like AZI1, is essential for priming defenses and is reported to phosphorylate AZI1 in vitro.To better understand the molecular basis of plant immunity, it is essential to assess the location and movement of chloroplast‐derived priming signals; here, I report on the function of AZI1’s localization to chloroplasts and the regulation of this chloroplast targeting using the model plants Arabidopsis thaliana and Nicotiana benthamiana. A more thorough understanding of priming will have profound agricultural benefits via genetic engineering or breeding of crops. Enhanced priming will boost immunity only during infection and would reduce fitness costs associated with constitutively active defenses.Support or Funding InformationThis work is supported by the NSF, the MCB training grant (T32 GM007183), the Ford Predoctoral Fellowship, and the HHMI Gilliam Fellowship for Advanced Study.