Search

Chloroplast-to-nucleus retrograde signaling (RS) pathways are critical in modulating plant development and stress adaptation. Among chloroplast proteins mediating RS pathways, GENOMES UNCOUPLED1 (GUN1) represses the transcription of the nuclear transcription factors GOLDEN2-LIKE1 (GLK1) and GLK2 that positively regulate chloroplast biogenesis. Given the extensive exploration of the function of GUN1 in biogenic RS carried out in previous years, our understanding of its role in plant stress responses remains scarce. Here, we revealed that GUN1 contributes to the expression of salicylic acid (SA)-responsive genes (SARGs) through transcriptional repression of GLK1/2 in Arabidopsis (Arabidopsis thaliana). Loss of GUN1 significantly compromised the SA responsiveness in plants, concomitant with the upregulation of GLK1/2 transcripts. In contrast, knockout of GLK1/2 potentiated the expression of SARGs and led to enhanced stress responses. Chromatin immunoprecipitation, coupled with quantitative PCR and related reverse genetic approaches, unveiled that in gun1, GLK1/2 might modulate SA-triggered stress responses by stimulating the expression of WRKY18 and WRKY40, transcriptional repressors of SARGs. In summary, we demonstrate that a hierarchical regulatory module, consisting of GUN1-GLK1/2-WRKY18/40, modulates SA signaling, opening a research avenue regarding a latent GUN1 function in plant–environment interactions.

Oxidative post-translational modifications of specific chloroplast proteins contribute to the initiation of retrograde signaling. The Arabidopsis thaliana EXECUTER1 (EX1) protein, a chloroplast-localized singlet oxygen (

In plants, RNA-directed DNA methylation (RdDM) is a well-known de novo DNA methylation pathway that involves two plant-specific RNA polymerases, Pol IV and Pol V. In this study, we discovered and characterized an RdDM factor, RDM15. Through DNA methylome and genome-wide siRNA analyses, we show that RDM15 is required for RdDM-dependent DNA methylation and siRNA accumulation at a subset of RdDM target loci. We show that RDM15 contributes to Pol V-dependent downstream siRNA accumulation and interacts with NRPE3B, a subunit specific to Pol V. We also show that the C-terminal tudor domain of RDM15 specifically recognizes the histone 3 lysine 4 monomethylation (H3K4me1) mark. Structure analysis of RDM15 in complex with the H3K4me1 peptide showed that the RDM15 tudor domain specifically recognizes the monomethyllysine through an aromatic cage and a specific hydrogen bonding network; this chemical feature-based recognition mechanism differs from all previously reported monomethyllysine recognition mechanisms. RDM15 and H3K4me1 have similar genome-wide distribution patterns at RDM15-dependent RdDM target loci, establishing a link between H3K4me1 and RDM15-mediated RdDM in vivo. In summary, we have identified and characterized a histone H3K4me1-specific binding protein as an RdDM component, and structural analysis of RDM15 revealed a chemical feature-based lower methyllysine recognition mechanism., In plants, RNA-directed DNA methylation (RdDM) is a de novo DNA methylation pathway that is responsible for transcriptional silencing of repetitive elements. Here, the authors characterized a new RdDM factor, RDM15, and show that it is required for RdDM-dependent DNA methylation and siRNA accumulation at a subset of RdDM target loci.

Homologous recombination-mediated gene targeting (GT) enables precise sequence knockin or sequence replacement, and thus is a powerful tool for heritable precision genome engineering. We recently established a clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9)-mediated approach for heritable GT in Arabidopsis (Arabidopsis thaliana), but its broad utility was not tested, and the underlying molecular mechanism was unclear. Here, we achieved precise GT at 14 out of 27 tested endogenous target loci using the sequential transformation approach and obtained vector-free GT plants by backcrossing. Thus, the sequential transformation GT method provides a broadly applicable technology for precise genome manipulation. We show that our approach generates heritable GT in the egg cell or early embryo of T1 Arabidopsis plants. Analysis of imprecise GT events suggested that single-stranded transfer DNA (T-DNA)/VirD2 complexes produced during the Agrobacterium (Agrobacterium tumefaciens) transformation process may serve as the donor templates for homologous recombination-mediated repair in the GT process. This study provides new insights into the molecular mechanisms of CRISPR/Cas9-mediated GT in Arabidopsis.

Significance Although plastid division is critical for plant development, how components of the plastid division machinery (PDM) are imported into plastids remains unexplored. A forward genetic screen to identify suppressors of a crumpled leaf ( crl ) mutant deficient in plastid division led us to find dominant gain-of-function (GF) mutations in TIC236 , which significantly increases the import of PDM components and completely rescues crl phenotypes. The defective plastid division phenotypes in crl and tic236-knockdown mutants and CRL-TIC236 association in a functional complex indicate that the CRL-TIC236 module is vital for plastid division. Hence, we report the first GF translocon mutants and unveil CRL as a novel functional partner of TIC236 for PDM import.

Clathrin-mediated vesicular formation and trafficking are highly conserved in eukaryotic cells and are responsible for molecular cargo transport and signal transduction among organelles. It remains largely unknown whether clathrin-coated vesicles can be generated from chloroplasts. CHLOROPLAST VESICULATION (CV)-containing vesicles (CVVs) generate from chloroplasts and mediate chloroplast degradation under abiotic stress. In this study, we showed that CV interacted with the clathrin heavy chain (CHC) and induced vesicle budding from the chloroplast inner envelope membrane. Defects on CHC2 and the dynamin-encoding DRP1A gene affected CVV budding and releasing from chloroplast. CHC2 is also required for CV-induced chloroplast degradation and hypersensitivity to water stress. Moreover, GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPC) interacts with CV and impairs the CV-CHC2 interaction. GAPC1 overexpression inhibited CV-mediated chloroplast degradation and hypersensitivity to water stress. CV silencing alleviated the hypersensitivity of gapc1gapc2 plant to water stress. Together, our work revealed a pathway of clathrin-assisted CVV budding from the chloroplast inner envelope membrane, which mediated the stress-induced chloroplast degradation and stress response.

Chloroplasts mediate genetically controlled cell death via chloroplast-to-nucleus retrograde signaling. To decipher the mechanism, we examined chloroplast-linked lesion-mimic mutants of Arabidopsis (Arabidopsis thaliana) deficient in plastid division, thereby developing gigantic chloroplasts (GCs). These GC mutants, including crumpled leaf (crl), constitutively express immune-related genes and show light-dependent localized cell death (LCD), mirroring typical autoimmune responses. Our reverse genetic approach excludes any potential role of immune/stress hormones in triggering LCD. Instead, transcriptome and in silico analyses suggest that reactive electrophile species (RES) generated via oxidation of polyunsaturated fatty acids (PUFAs) or lipid peroxidation-driven signaling may induce LCD. Consistent with these results, the one of the suppressors of crl, dubbed spcrl4, contains a causative mutation in the nuclear gene encoding chloroplast-localized FATTY ACID DESATURASE5 (FAD5) that catalyzes the conversion of palmitic acid (16:0) to palmitoleic acid (16:1). The loss of FAD5 in the crl mutant might attenuate the levels of RES and/or lipid peroxidation due to the reduced levels of palmitic acid-driven PUFAs, which are prime targets of reactive oxygen species. The fact that fad5 also compromises the expression of immune-related genes and the development of LCD in other GC mutants substantiates the presence of an intrinsic retrograde signaling pathway, priming the autoimmune responses in a FAD5-dependent manner.

The plant stress hormone salicylic acid (SA) participates in local and systemic acquired resistance, which eventually leads to whole-plant resistance to bacterial pathogens. However, if SA-mediated signaling is not appropriately controlled, plants incur defense-associated fitness costs such as growth inhibition and cell death. Despite its importance, to date only a few components counteracting the SA-primed stress responses have been identified in Arabidopsis (Arabidopsis thaliana). These include other plant hormones such as jasmonic acid and abscisic acid, and proteins such as LESION SIMULATING DISEASE1, a transcription coregulator. Here, we describe PLANT NATRIURETIC PEPTIDE A (PNP-A), a functional analog to vertebrate atrial natriuretic peptides, that appears to antagonize the SA-mediated plant stress responses. While loss of PNP-A potentiates SA-mediated signaling, exogenous application of synthetic PNP-A or overexpression of PNP-A significantly compromises the SA-primed immune responses. Moreover, we identify a plasma membrane–localized receptor-like protein, PNP-R2, that interacts with PNP-A and is required to initiate the PNP-A–mediated intracellular signaling. In summary, our work identifies a peptide and its putative cognate receptor as counteracting both SA-mediated signaling and SA-primed cell death in Arabidopsis.

GOLDEN2-LIKE (GLK) transcription factors drive the expression of photosynthesis-associated nuclear genes (PhANGs) indispensable for chloroplast biogenesis. Salicylic acid (SA)-induced SIGMA FACTOR-BINDING PROTEIN 1 (SIB1), a transcription coregulator and positive regulator of cell death, interacts with GLK1 and GLK2 to reinforce the expression of PhANGs, leading to photoinhibition of photosystem II and singlet oxygen (1O2) burst in chloroplasts. 1O2 then contributes to SA-induced cell death via EXECUTER 1 (EX1; 1O2 sensor protein)-mediated retrograde signaling upon reaching a critical level. This earlier finding has initiated research on the potential role of GLK1/2 and EX1 in SA signaling. Consistent with this view, we reveal that LESION-SIMULATING DISEASE 1 (LSD1), a transcription coregulator and negative regulator of SA-primed cell death, interacts with GLK1/2 to repress their activities in Arabidopsis (Arabidopsis thaliana). Overexpression of LSD1 repressed GLK target genes, including PhANGs, whereas loss of LSD1 enhanced their expression. Remarkably, LSD1 overexpression inhibited chloroplast biogenesis, resembling the characteristic glk1glk2 double mutant phenotype. Subsequent chromatin immunoprecipitation coupled with expression analyses further revealed that LSD1 inhibits the DNA-binding activity of GLK1 toward its target promoters. SA-induced nuclear-targeted SIB1 proteins appeared to interrupt the LSD1–GLK interaction, and the subsequent SIB1–GLK interaction activated EX1-mediated 1O2 signaling, elucidating antagonistic modules SIB1 and LSD1 in the regulation of GLK activity. Taken together, we provide a working model that SIB1 and LSD1, mutually exclusive SA-signaling components, antagonistically regulate GLK1/2 to fine-tune the expression of PhANGs, thereby modulating 1O2 homeostasis and related stress responses.

Low and high temperatures are life-threatening stress factors, diminishing plant productivity. One of the earliest responses of plants to stress is a rapid burst of reactive oxygen species (ROS) in chloroplasts. Widespread efforts over the past decade shed new light on the chloroplast as an environmental sensor, translating the environmental fluctuation into varying physiological responses by utilizing distinct retrograde (chloroplast-to-nucleus) signals. Recent studies have unveiled that chloroplasts mediate a similar unfolded/misfolded/damaged protein response (cpUPR) as observed in the endoplasmic reticulum and mitochondria. Although observing cpUPR is not surprising since the chloroplast is a prime organelle producing harmful ROS, the intertwined relationship among ROS, protein damage, and chloroplast protein quality controls (cpPQCs) with retrograde signaling has recently been reported. This finding also gives rise to critical attention on chloroplast proteins involved in cpPQCs, ROS detoxifiers, transcription/translation, import of precursor proteins, and assembly/maturation, the deficiency of which compromises chloroplast protein homeostasis (proteostasis). Any perturbation in the protein may require readjustment of proteostasis by transmitting retrograde signal(s) to the nucleus, whose genome encodes most of the chloroplast proteins involved in proteostasis. This review focuses on recent findings on cpUPR and chloroplast-targeted FILAMENTOUS TEMPERATURE-SENSITIVE H proteases involved in cpPQC and retrograde signaling and their impacts on plant responses to temperature stress.

Summary We present a protocol for analyzing and evaluating the relocalization of proteins from the plasma membrane to chloroplasts. Some plant membrane-bound proteins carry dual targeting signals, e.g., a membrane-anchoring N-myristoylation motif and a chloroplast transit peptide. These proteins are predominantly targeted to membranes; upon certain cues, however, they can undergo detachment from membranes and relocalization to chloroplasts. This protocol combines imaging and biochemical analyses to track in a reliable and quantitative manner the relocalization of proteins between subcellular organelles. For complete details on the use and execution of this protocol, please refer to Medina-Puche et al. (2020)., Graphical abstract, Highlights • A method for tracking relocation of proteins from plasma membrane-to-chloroplasts • A method for a quantitative evaluation of the localization in a dynamic manner • Relocation of both host and foreign proteins can be studied • The protocol integrates biochemical and imaging approaches, We present a protocol for analyzing and evaluating the relocalization of proteins from the plasma membrane to chloroplasts. Some plant membrane-bound proteins carry dual targeting signals, e.g., a membrane-anchoring N-myristoylation motif and a chloroplast transit peptide. These proteins are predominantly targeted to membranes; upon certain cues, however, they can undergo detachment from membranes and relocalization to chloroplasts. This protocol combines imaging and biochemical analyses to track in a reliable and quantitative manner the relocalization of proteins between subcellular organelles.

SummaryThe chloroplast translocons TOC75 and TIC236 are homologs of the bacterial translocation and assembly module (Tam) A and TamB involved in protein export. Here, we unveil a TIC236-allied component, the chloroplast outer membrane protein CRUMPLED LEAF (CRL), absence of which impairs plastid division and induces autoimmune responses in Arabidopsis thaliana. A forward genetic screen aimed at finding crl suppressors revealed multiple TIC236 gain-of-function mutations (TIC236GFs). Despite the low sequence identity between TIC236 and bacterial TamB, each mutated TIC236GF residue is conserved in TamB. Consistently, a tic236- knockdown mutant exhibited multiple lesion phenotypes similar to crl, indicating a shared functionality of CRL and TIC236. Ensuing reverse genetic analyses revealed genetic interaction between CRL and SP1, a RING-type ubiquitin E3 ligase, as well as with the plastid protease FTSH11, which function in TOC and TIC protein turnover, respectively. Loss of either SP1 or FTSH11 rescued crl mutant phenotypes to varying degrees due to increased translocon levels. Consistent with impaired plastid division exhibited by both crl and tic236-knockdown mutants, CRL interacts with the transit peptides of proteins essential in plastid division, and TIC236GF mutant proteins reinforce their import via increased TIC236 stability. Overall, our data shed new light on the links between plastid division, plant stress response and plastid protein import. We have also isolated and characterized the first GF mutants exhibiting increased protein import efficiency, which may inspire chloroplast engineering for agricultural advancement.

Environmental information perceived by chloroplasts can be translated into retrograde signals that alter the expression of nuclear genes. Singlet oxygen (1O2) generated by photosystem II (PSII) can cause photo-oxidative damage of PSII but has also been implicated in retrograde signaling. We previously reported that a nuclear-encoded chloroplast FtsH2 metalloprotease coordinates 1O2-triggered retrograde signaling by promoting the degradation of the EXECUTER1 (EX1) protein, a putative 1O2 sensor. Here, we show that a 1O2-mediated oxidative post-translational modification of EX1 is essential for initiating 1O2-derived signaling. Specifically, the Trp643 residue in DUF3506 domain of EX1 is prone to oxidation by 1O2. Both the substitution of Trp643 with 1O2-insensitive amino acids and the deletion of the DUF3506 domain abolish the EX1-mediated 1O2 signaling. We thus provide mechanistic insight into how EX1 senses 1O2 via Trp643 located in the DUF3506 domain., Singlet oxygen generated by photosynthesis under photo-oxidative stress conditions triggers retrograde signaling from plastids to nuclei. Here, the authors show that singlet oxygen perception and subsequent signaling events require oxidative post-translational modification of the EXECUTER1 protein.

Arabidopsis var2 mutants with defective PSII repair trigger chloroplast-to-nucleus retrograde signaling that results in a response resembling the unfolded protein response, leading to accumulation of proteins involved in proteostasis and detoxification., Cellular protein homeostasis (proteostasis) is maintained through the balance between de novo synthesis and proteolysis. The unfolded/misfolded protein response (UPR) that is triggered by stressed endoplasmic reticulum (ER) also plays an important role in proteostasis in both plants and animals. Although ER-triggered UPR has been extensively studied in plants, the molecular mechanisms underlying mitochondrial and chloroplastic UPRs are largely uncharacterized despite the fact that these organelles are sites of production of harmful reactive oxygen species (ROS), which damage proteins. In this study, we demonstrate that chloroplasts of the Arabidopsis yellow leaf variegation 2 (var2) mutant, which lacks the metalloprotease FtsH2, accumulate damaged chloroplast proteins and trigger a UPR-like response, namely the accumulation of a suite of chloroplast proteins involved in protein quality control (PQC). These PQC proteins include heat-shock proteins, chaperones, proteases, and ROS detoxifiers. Given that FtsH2 functions primarily in photosystem II proteostasis, the accumulation of PQC-related proteins may balance the FtsH2 deficiency. Moreover, the apparent up-regulation of the cognate transcripts indicates that the accumulation of PQC-related proteins in var2 is probably mediated by retrograde signaling, indicating the occurrence of a UPR-like response in var2.

In this paper, we consider a signal-level maximum likelihood detection for multiple-input multiple-output filter-bank multicarrier-quadrature amplitude modulation (MIMO FBMC-QAM) system with two prototype filters. Under high-frequency selective channels, the conventional symbol-level maximum likelihood detection suffers from degraded bit error rate performances because it cannot utilize the oversampling characteristics of the signal-level. However, due to intrinsic interference and high computational complexity, a maximum likelihood detection cannot be applied directly to the signal-level. To solve this problem, we propose a signal-level maximum likelihood detection based on partial candidates. Utilizing the diagonal dominant characteristics of effective channels, the proposed signal-level maximum likelihood detection can reduce the effect of intrinsic interference with practical computational complexity. Moreover, a region ordering algorithm based on Euclidean distance can improve the efficiency of the proposed signal-level maximum likelihood detection. Simulation results show that the proposed signal-level maximum likelihood detection can outperform the bit error rate performance of the conventional symbol-level maximum likelihood detection.

Chloroplast-to-nucleus retrograde signaling is essential for the coupled expression of photosynthesis-associated nuclear genes (PhANGs) and plastid genes (PhAPGs) to ensure the functional status of chloroplasts (Cp) in plants. Although various signaling components involved in the process have been identified in Arabidopsis (Arabidopsis thaliana), the biological relevance of such coordination remains an enigma. Here, we show that the uncoupled expression of PhANGs and PhAPGs contributes to the cell death in the lesion simulating disease1 (lsd1) mutant of Arabidopsis. A daylength-dependent increase of salicylic acid (SA) appears to rapidly up-regulate a gene encoding SIGMA FACTOR BINDING PROTEIN1 (SIB1), a transcriptional coregulator, in lsd1 before the onset of cell death. The dual targeting of SIB1 to the nucleus and the Cps leads to a simultaneous up-regulation of PhANGs and down-regulation of PhAPGs. Consequently, this disrupts the stoichiometry of photosynthetic proteins, especially in PSII, resulting in the generation of the highly reactive species singlet oxygen ((1)O(2)) in Cps. Accordingly, inactivation of the nuclear-encoded Cp protein EXECUTER1, a putative (1)O(2) sensor, significantly attenuates the lsd1-conferred cell death. Together, these results provide a pathway from the SA- to the (1)O(2)-signaling pathway, which are intertwined via the uncoupled expression of PhANGs and PhAPGs, contributing to the lesion-mimicking cell death in lsd1.

In this paper, the problem of reducing the peak-to-average power ratio (PAPR) of a discrete Fourier transform spread orthogonal frequency-division multiplexing signal is considered for the transmission of BPSK symbols. In particular, a joint optimization of the constellation-rotation angle and the pulse-shaping vector is conducted with or without the allocation of additional sub-carriers. To avoid the exponential complexity required to evaluate the PAPR, a computationally efficient upper bound on the worst-case PAPR is derived and adopted as the objective function. Unlike conventional upper bounds, the proposed upper bound exploits the structure in the relative phase among symbol waveforms and incorporates the constellation-rotation effect. The signal-to-interference-plus-noise ratio (SINR) at the output of a constellation-derotating matched filter is also derived along with a generalized Nyquist criterion and adopted as the inequality constraint function. Then, joint optimizations are conducted through numerical search for various signal parameters and target SINRs. Surprisingly, the optimal rotation angle is neither 0 nor $\pi /2$ . It is shown that the proposed combinations significantly outperform the existing combinations of the rotation angle and the shaping vector when the signal is amplified by a nonlinear power amplifier.

GOLDEN2-LIKE (GLK) transcription factors drive the expression of photosynthesis-associated nuclear genes (PhANGs), indispensable for chloroplast biogenesis. We previously demonstrated that the salicylic acid (SA)-induced SIGMA FACTOR-BINDING PROTEIN 1 (SIB1), a transcription coregulator and positive regulator of cell death, interacts with GLK1 and GLK2 to reinforce their activities. The SIB1-GLK interaction raises the level of light-harvesting antenna proteins in photosystem II, aggravating photoinhibition and singlet oxygen (1O2) burst. 1O2 then contributes to SA-induced cell death via EXECUTER 1 (EX1, 1O2 sensor protein)-mediated retrograde signaling upon reaching a critical level. We now reveal that LESION-SIMULATING DISEASE 1 (LSD1), a transcription coregulator and negative regulator of SA-primed cell death, interacts with GLK1/2 to repress their activities. Consistently, the overexpression of LSD1 represses GLK target genes including PhANGs, whereas the loss of LSD1 increases their expression. Remarkably, LSD1 overexpression inhibits chloroplast biogenesis, resembling the characteristic glk1glk2 double mutant phenotype. The subsequent chromatin immunoprecipitation analysis coupled with quantitative PCR further reveals that LSD1 inhibits the DNA-binding activity of GLK1 towards its target promoters. The SA-induced nuclear-targeted SIB1 appears to counteractively interact with GLK1/2, leading to the activation of EX1-mediated 1O2 signaling. Taken together, we provide a working model that SIB1 and LSD1, mutually exclusive SA-signaling components, antagonistically regulate GLK1/2 to fine-tune the expression of PhANGs, thereby modulating 1O2 homeostasis and related stress responses.

In plants, RNA-directed DNA methylation (RdDM) is a well-known de novo DNA methylation pathway that involves two plant-specific RNA polymerases, Pol IV and Pol V. In this study, we discovered and characterized a new RdDM factor, RDM15. Through DNA methylome and genome wide siRNA analyses, we show that RDM15 is required for RdDM-dependent DNA methylation and siRNA accumulation at a subset of RdDM target loci. We show that RDM15 contributes to Pol V-dependent downstream siRNA accumulation, and interacts with NRPE3B, a subunit specific to Pol V. We also show that the C-terminal tudor domain of RDM15 specifically recognizes the histone 3 lysine 4 monomethylation (H3K4me1) mark. Structure analysis of RDM15 in complex with the H3K4me1 peptide showed that the RDM15 tudor domain specifically recognizes the monomethyllysine through an aromatic cage and a specific hydrogen bonding network; this chemical feature-based recognition mechanism differs from all previously reported lower methyllysine recognition mechanisms. RDM15 and H3K4me1 have similar genome-wide distribution patterns at RDM15-dependent RdDM target loci, establishing a link between H3K4me1 and RDM15-mediated RdDM in vivo. In summary, we have identified and characterized a histone H3K4me1-specific binding protein as a new RdDM component, and our structural analysis of RDM15 revealed a new type of chemical feature-based lower methyllysine recognition mechanism.

Flowering plants sense various environmental and endogenous signals to trigger the floral transition and start the reproductive growth cycle. CONSTANS (CO) is a master transcription factor in the photoperiod floral pathway that integrates upstream signals and activates the florigen gene FLOWERING LOCUS T (FT). Here, we performed comprehensive structural and biochemical analyses to study the molecular mechanism underlying the regulation of FT by CO in Arabidopsis thaliana. We show that the four previously characterized cis-elements in the FT promoter proximal region, CORE1, CORE2, P1, and P2, are all direct CO binding sites. Structural analysis of CO in complex with NUCLEAR FACTOR-YB/YC (NF–YB/YC) and the CORE2 or CORE1 elements revealed the molecular basis for the specific recognition of the shared TGTG motifs. Biochemical analysis suggested that CO might form a homomultimeric assembly via its N-terminal B-Box domain and simultaneously occupy multiple cis-elements within the FT promoter. We suggest that this multivalent binding gives the CO–NF–Y complex high affinity and specificity for FT promoter binding. Overall, our data provide a detailed molecular model for the regulation of FT by the master transcription factor complex CO–NF–Y during the floral transition.

Chloroplasts overproduce reactive oxygen species (ROS) under unfavorable environmental conditions, and these ROS are implicated in both signaling and oxidative damage. There is mounting evidence for their roles in translating environmental fluctuations into distinct physiological responses, but their targets, signaling cascades, and mutualism and antagonism with other stress signaling cascades and within ROS signaling remain poorly understood. Great efforts made in recent years have shed new light on chloroplast ROS-directed plant stress responses, from ROS perception to plant responses, in conditional mutants of Arabidopsis thaliana or under various stress conditions. Some articles have also reported the mechanisms underlying the complexity of ROS signaling pathways, with an emphasis on spatiotemporal regulation. ROS and oxidative modification of affected target proteins appear to induce retrograde signaling pathways to maintain chloroplast protein quality control and signaling at a whole-cell level using stress hormones. This review focuses on these seemingly interconnected chloroplast-to-nucleus retrograde signaling pathways initiated by ROS and ROS-modified target molecules. We also discuss future directions in chloroplast stress research to pave the way for discovering new signaling molecules and identifying intersectional signaling components that interact in multiple chloroplast signaling pathways., Chloroplast-derived reactive oxygen species (ROS) cause oxidative damage to macromolecules. However, oxidation-/damage-associated signaling pathways and their interplay with other signaling pathways remain largely unexplored. This review focuses mainly on chloroplast-mediated signaling pathways elicited by ROS and ROS-modified target molecules and discusses their potential interplay with other known stress-related signaling pathways.

N-terminal (Nt) acetylation (NTA) is an ample and irreversible cotranslational protein modification catalyzed by ribosome-associated Nt-acetyltransferases. NTA on specific proteins can act as a degradation signal (called an Ac/N-degron) for proteolysis in yeast and mammals. However, in plants, the biological relevance of NTA remains largely unexplored. In this study, we reveal that Arabidopsis (Arabidopsis thaliana) SIGMA FACTOR-BINDING PROTEIN1 (SIB1), a transcription coregulator and a positive regulator of salicylic acid-primed cell death, undergoes an absolute NTA on the initiator Met; Nt-acetyltransferase B (NatB) partly contributes to this modification. While NTA results in destabilization of certain target proteins, our genetic and biochemical analyses revealed that plant NatB-involved NTA instead renders SIB1 more stable. Given that the ubiquitin/proteasome system stimulates SIB1 degradation, it seems that the NTA-conferred stability ensures the timely expression of SIB1-dependent genes, mostly related to immune responses. Taking our findings together, here we report a noncanonical NTA-driven protein stabilization in land plants.

Singlet oxygen (1O2) is an excited state of molecular oxygen with an electron spin shift in the molecular orbitals, which is extremely unstable and highly reactive. In plants, 1O2 is primarily generated as a byproduct of photosynthesis in the photosystem II reaction center (PSII RC) and the light-harvesting antenna complex (LHC) in the grana core (GC). This occurs upon the absorption of light energy when the excited chlorophyll molecules in the PSII transfer the excess energy to molecular oxygen, thereby generating 1O2. As a potent oxidant, 1O2 promotes oxidative damage. However, at sub-lethal levels, it initiates chloroplast-to-nucleus retrograde signaling to contribute to plant stress responses, including acclimation and cell death. The thylakoid membranes comprise two spatially separated 1O2 sensors: β-carotene localized in the PSII RC in the GC and the nuclear-encoded chloroplast protein EXECUTER1 (EX1) residing in the non-appressed grana margin (GM). Finding EX1 in the GM suggests the existence of an additional source of 1O2 in the GM and the presence of two distinct 1O2-signaling pathways. In this review, we mainly discuss the genesis and impact of 1O2 in plant physiology.

SUMMARYChloroplasts are crucial players in the activation of defensive hormonal responses during plant-pathogen interactions. Here, we show that a plant virus-encoded protein re-localizes from the plasma membrane to chloroplasts upon triggering plant defence, interfering with the chloroplast-dependent activation of anti-viral salicylic acid (SA) biosynthesis. Strikingly, we have found that plant pathogens from different kingdoms seem to have convergently evolved to target chloroplasts and impair SA-dependent defences following an association with membranes, which is based on the co-existence of two subcellular targeting signals, an N-myristoylation site and a chloroplast transit peptide. This pattern is also present in plant proteins, at least one of which conversely activates SA defences from the chloroplast. Taken together, our results suggest that a pathway linking plasma membrane to chloroplasts and activating defence exists in plants, and that such pathway has been co-opted by plant pathogens during host-pathogen co-evolution to promote virulence through suppression of SA responses.

Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are an important microbe-associated molecular pattern (MAMP) that triggers immune responses in plants and animals. A previous genetic screen in Arabidopsis (Arabidopsis thaliana) identified LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE), a B-type lectin S-domain receptor kinase, as a sensor of LPS. However, the LPS-activated LORE signaling pathway and associated immune responses remain largely unknown. In this study, we found that LPS trigger biphasic production of reactive oxygen species (ROS) in Arabidopsis. The first transient ROS burst was similar to that induced by another MAMP, flagellin, whereas the second long-lasting burst was induced only by LPS. The LPS-triggered second ROS burst was found to be conserved in a variety of plant species. Microscopic observation of the generation of ROS revealed that the LPS-triggered second ROS burst was largely associated with chloroplasts, and functional chloroplasts were indispensable for this response. The lipid A moiety, the most conserved portion of LPS, appears to be responsible for the second ROS burst. Surprisingly, the LPS- and lipid A-triggered second ROS burst was only partially dependent on LORE. Together, our findings provide insight on the LPS-triggered ROS production and the associated signaling pathway.

The asynchronous heterogeneous network scenario is becoming one of the key features for 5G mobile communications. Compared to CP-OFDM, which is 4G waveform, superior spectrum confinement as well as higher spectral efficiency has been taken into consideration for 5G waveform design. In this article, we introduce a new waveform called QAM-FBMC that provides superior spectrum confinement and higher spectral efficiency simultaneously against CP-OFDM. The article explains fundamental trade-offs in waveform design and shows how they can be optimized for QAM-FBMC. We also introduce the transmission and reception procedures for QAM-FBMC including its similarities and differences compared to well-known waveforms such as CP-OFDM and OQAM-FBMC. Performance evaluation results show where the spectral efficiency gain of QAM-FBMC over CP-OFDM comes from.

Image steganography hides secret information in the cover images so naturally that the existence of hidden data in the stego-image is not recognisable. This Letter proposes a new approach to blind decoding of image steganography using the local entropy distributions of decoded images. The local entropy distributions of incorrectly decoded images are different from those of normal ones because of the abnormal image structures in the erroneously decoded images. This blind decoding in the image steganography is very useful to extract hidden image information because there are enormous least significant bit (LSB)-based steganography methods, and it is very hard to find the methods by observing manipulated LSBs.

Singlet oxygen ((1)O(2)) is a potent oxidizing agent, principally generated by photosystem II (PSII) as a byproduct of photosynthesis. Hence, (1)O(2) damages PSII, especially the PSII reaction center (RC) proteins, promoting a process called PSII repair cycle. The hetero-hexameric FtsH protease, located in the thylakoid membrane, is essential in degrading these damaged PSII RC proteins, which defines the first step of the PSII repair. The loss of the central subunit of the FtsH protease, FtsH2 (VAR2), weakens the PSII repair, thereby impairing PSII proteostasis. A recent study demonstrated that the impaired proteostasis (or accumulation of damaged proteins) in the chloroplasts of the var2 mutant induces an unfolded/misfolded protein response (UPR)-like response, more appropriately referred to as a damaged protein response (DPR), as evident in the accumulation of proteins related to the protein quality control (PQC). Comparison of data from chloroplast proteomics data with RNA sequencing in the context of the UPR-like response suggests a plausible activation of retrograde signaling in the var2 mutant. Either through the enhanced level of (1)O(2) or by impairing the substrate-unfolding activity of FtsH2, the reinforced defect appears to induce stress-related genes via the stress hormone salicylic acid (SA). This finding suggests that impaired chloroplast proteostasis (specifically for PSII proteins) may activate the chloroplast-established isochorismate pathway to produce SA. If this assumption is correct, then SA serves as a retrograde signaling molecule. In this review, we will discuss the impact of chloroplast proteostasis on chloroplasts-to-nucleus communication.

Peptide hormones perceived in the cell surface via receptor proteins enable cell-to-cell communication and act in multiple biological processes through the activation of intracellular signaling. Even though Arabidopsis is predicted to have more than 1,000 secreted peptides, the biological relevance of the majority of these is yet to be established. Here, we demonstrate that PLANT NATRIURETIC PEPTIDE A (PNP-A), a functional analog to vertebrate atrial natriuretic peptides, antagonizes the salicylic acid (SA)-mediated cell death in the Arabidopsis lesion-stimulating disease 1 (lsd1) mutant. While loss of PNP-A potentiates SA signaling, exogenous application of the PNP-A synthetic peptide or overexpression of PNP-A significantly compromises the SA-mediated cell death. Moreover, we identified a plasma membrane-localized receptor-like protein, which we name PNPAR (for PNP-A receptor), that binds PNP-A and is required to counteract SA responses. Our work identifies a novel peptide-receptor pair which modulates SA responses in Arabidopsis.

Chloroplasts are crucial players in the activation of defensive hormonal responses during plant-pathogen interactions. Here, we show that a plant virus-encoded protein re-localizes from the plasma membrane to chloroplasts upon activation of plant defense, interfering with the chloroplast-dependent anti-viral salicylic acid (SA) biosynthesis. Strikingly, we have found that plant pathogens from different kingdoms seem to have convergently evolved to target chloroplasts and impair SA-dependent defenses following an association with membranes, which relies on the co-existence of two subcellular targeting signals, an N-myristoylation site and a chloroplast transit peptide. This pattern is also present in plant proteins, at least one of which conversely activates SA defenses from the chloroplast. Taken together, our results suggest that a pathway linking plasma membrane to chloroplasts and activating defense exists in plants and that such pathway has been co-opted by plant pathogens during host-pathogen co-evolution to promote virulence through suppression of SA responses.

Photosystem II reaction center (PSII RC) and light-harvesting complex (LHC) inevitably generate highly reactive singlet oxygen (1O2) that can impose photo-oxidative damage, especially when the rate of generation exceeds the rate of detoxification. Besides being toxic, 1O2 has also been ascribed to trigger retrograde signaling, which leads to nuclear gene expression changes. Two distinctive molecular components appear to regulate 1O2 signaling: a volatile signaling molecule s-cyclocitral (s-CC) generated upon oxidation of s-carotene by 1O2 in PSII RC assembled in grana core, and a thylakoid membrane-bound FtsH2 metalloprotease that promotes 1O2-triggered signaling through the proteolysis of EXECUTER1 (EX1) proteins associated with PSII in grana margin. The role of FtsH2 protease in 1O2 signaling was established recently in the conditional fluorescent (flu) mutant of Arabidopsis thaliana that generates 1O2 upon dark-to-light shift. The flu mutant lacking functional FtsH2 significantly impairs 1O2-triggered and EX1-mediated cell death. In the present study, the role of FtsH2 in the induction of 1O2 signaling was further clarified by analyzing the FtsH2-dependent nuclear gene expression changes in the flu mutant. Genome-wide transcriptome analysis showed that the inactivation of FtsH2 repressed the majority (85%) of the EX1-dependent 1O2-responsive genes (ESORGs), providing direct connection between FtsH2-mediated EX1 degradation and 1O2-triggered gene expression changes. Furthermore, the overlap between β-CC-induced genes and EX1-FtsH2-dependent genes was very limited, further supporting the coexistence of two distinctive 1O2 signaling pathways.

Singlet oxygen ((1)O2)-mediated signaling has been established in the conditional fluorescent (flu) mutant of Arabidopsis. In the dark, the flu mutant accumulates free protochlorophyllide (Pchlide), a photosensitizer that in the light generates (1)O2. The release of (1)O2 leads to growth inhibition of mature plants and bleaching of seedlings. These (1)O2-mediated responses depend on two plastid proteins, EXECUTER (EX) 1 and 2. An ex1/ex2/flu mutant accumulates in the dark Pchlide and upon illumination generates similar amounts of (1)O2 as flu, but (1)O2-mediated responses are abrogated in the triple mutant. The (1)O2- and EX-dependent signaling pathway operates also in wild type placed under light stress. However, it does not act alone as in flu, but interacts with other signaling pathways that modulate (1)O2-mediated responses. Depending on how severe the light stress is, (1)O2- and EX-dependent signaling may be superimposed by (1)O2-mediated signaling that does not depend on EX and is associated with photo-oxidative damage. Because of its high reactivity and short half-life, (1)O2 is unlikely to be a signal that is translocated across the chloroplast envelope, but is likely to interact with other plastid components close to its site of production and to generate more stable signaling molecules during this interaction. Depending on the site of (1)O2 production and the severity of stress, different signaling molecules may be expected that give rise to different (1)O2-mediated responses.

Significance Singlet oxygen ( 1 O 2 )- and EXECUTER1 (EX1)-dependent signaling triggers programmed cell death in seedlings and inhibits growth of mature plants of the fluorescent ( flu ) mutant of Arabidopsis . The EX1 protein has been located in chloroplasts to the grana margins close to where chlorophyll is synthesized and the disassembly of damaged photosystem II (PSII) and reassembly of active PSII take place. With the onset of 1 O 2 -mediated signaling there is a rapid decline of EX1 that depends on the ATP-dependent zinc metalloprotease FtsH. Generation of 1 O 2 without the decline of EX1 is not sufficient to activate 1 O 2 signaling. As FtsH cleaves also the D1 reaction center protein of damaged PSII, EX1-dependent signaling seems not only spatially but also functionally linked to the repair of PSII.

Retrograde plastid-to-nucleus signaling tightly controls and coordinates the nuclear and plastid gene expression that is required for plastid biogenesis and chloroplast activity. As chloroplasts act as sensors of environmental changes, plastid-derived signaling also modulates stress responses of plants by transferring stress-related signals and altering nuclear gene expression. Various mutant screens have been undertaken to identify constituents of plastid signaling pathways. Almost all mutations identified in these screens target plastid-specific but not extraplastidic functions. They have been suggested to define either genuine constituents of retrograde signaling pathways or components required for the synthesis of plastid signals. Here we report the characterization of the constitutive activator of AAA-ATPase (caa33) mutant, which reveals another way of how mutations that affect plastid functions may modulate retrograde plastid signaling. caa33 disturbs a plastid-specific function by impeding plastid division, and thereby perturbing plastid homeostasis. This results in preconditioning plants by activating the expression of stress genes, enhancing pathogen resistance and attenuating the capacity of the plant to respond to plastid signals. Our study reveals an intimate link between chloroplast activity and the susceptibility of the plant to stress, and emphasizes the need to consider the possible impact of preconditioning on retrograde plastid-to-nucleus signaling.

Link Adaptation should select the best modulation and coding scheme (MCS) which gives the highest throughput as channel conditions vary. Several link adaptation algorithms for wireless local area network (WLAN) have been proposed but for the future WLAN systems such as 802.11n system, these algorithms do not guarantee the best performance. In this paper, we propose a new link adaptation algorithm in which an MCS level is chosen by the received SNR plus the offset value obtained from the transmission results. The performance of proposed algorithm is simulated by an IEEE 802.11n system. From the analysis, we conclude the proposed algorithm performs better than the well-known link adaptation algorithms such as auto rate fallback and general SNR-based techniques. Particularly, the proposed algorithm improves throughput when the packet error ratio (PER) is constrained for fast fading channels.

In this letter, we analyze the post-scheduling SINR mismatch of a multiuser orthogonal random beamforming (ORBF) system. The mismatch probability is derived for a Gaussian broadcast channel with M transmit antennas and K single-antenna users. Numerical results are shown to be identical to theoretical results. The probability analysis enables us to calculate the minimum number of users to satisfy a target mismatch probability. Another way to use the mismatch probability is to control CQI mismatch handling mechanisms.

Shortly after the release of singlet oxygen ( 1 O 2 ), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of 1 O 2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of 1 O 2 -induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of 1 O 2 -dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1 / ex2 / flu triple mutant is sufficient to suppress the up-regulation of almost all 1 O 2 -responsive genes. Retrograde control of 1 O 2 -responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.