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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.

Chloroplast pre-ribosomal RNA (rRNA) undergoes maturation, which is critical for ribosome assembly. While the central and auxiliary factors in rRNA maturation have been elucidated in bacteria, their mode of action remains largely unexplored in chloroplasts. We now reveal chloroplast-specific factors involved in 16S rRNA maturation, RsmD methyltransferase (AtRsmD) and ribosome maturation factor RimM-like protein (AtRimM) in Arabidopsis thaliana. A forward genetic screen aimed to find suppressors of the Arabidopsis yellow variegated 2 (var2) mutant defective in photosystem II (PSII) quality control found a causal nonsense mutation in AtRsmD. The substantially impaired 16S rRNA maturation and translation due to the mutation rescued the leaf variegation phenotype by lowering the levels of PSII core proteins in var2. The subsequent co-immunoprecipitation coupled with mass spectrometry analyses and bimolecular fluorescence complementation assay found that AtRsmD interacts with AtRimM. Consistent with their interaction, loss of AtRimM also considerably impairs 16S rRNA maturation, with less methylation in m2G915 in 16S rRNA catalyzed by AtRsmD. The atrimM mutation also rescued var2 mutant phenotypes, corroborating the functional interplay between AtRsmD and AtRimM towards 16S rRNA maturation and chloroplast proteostasis.

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.

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.

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.

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.

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.

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.

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.

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.

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.