Retrograde signaling in plants: A critical review focusing on the GUN pathway and beyond

Plastids communicate their developmental and physiological status to the nucleus via retrograde signaling,allowing nuclear gene expression to be adjusted appropriately.Signaling during plastid biogenesis and responses of mature chloroplasts to environmental changes are designated“biogenic”and“operational”controls,respectively.A prominent example of the investigation of biogenic signaling is the screen for gun(genomes uncoupled)mutants.Although the first five gun mutants were identified 30 years ago,the functions of GUN proteins in retrograde signaling remain controversial,and that of GUN1 is hotly disputed.Here,we provide background information and critically discuss recently proposed concepts that address GUN-related signaling and some novel gun mutants.Moreover,considering heme as a candidate in retrograde signaling,we revisit the spatial organization of heme biosynthesis and export from plastids.Although this review focuses on GUN pathways,we also highlight recent progress in the identification and elucidation of chloroplast-derived signals that regulate the acclimation response in green algae and plants.Here,stress-induced accumulation of unfolded/misassembled chloroplast proteins evokes a chloroplast-specific unfolded protein response,which leads to changes in the expression levels of nucleus-encoded chaperones and proteases to restore plastid protein homeostasis.We also address the importance of chloroplast-derived signals for activation of flavonoid biosynthesis leading to production of anthocyanins during stress acclimation through sucrose non-fermenting 1-related protein kinase 1.Finally,a framework for identification and quantification of intercompartmental signaling cascades at the proteomic and metabolomic levels is provided,and we discuss future directions of dissection of organelle-nucleus communication.

Enhancing the light reactions of photosynthesis:Strategies,controversies,and perspectives

Photosynthesis is central to life on Earth,employing sunlight,water,and carbon dioxide to produce chemical energy and oxygen.It is generally accepted that boosting its efficiency offers one promising way to increase crop yields under agronomically realistic conditions.Since the components,structure,and regulatory mechanisms of the light reactions of photosynthesis are well understood,concepts for enhancing the process have been suggested and partially tested.These approaches vary in complexity,from targeting single components to comprehensive redesign of the whole process on the scales from single cells or tissues to whole canopies.Attempts to enhance light utilization per leaf,by decreasing pigmentation,increasing levels of photosynthetic proteins,prolonging the lifespan of the photosynthetic machinery,or massive reconfiguration of the photosynthetic machinery and the incorporation of nanomaterials,are discussed in this review first.Secondly,strategies intended to optimize the acclimation of photosynthesis to changes in the environment are presented,including redesigning mechanisms to dissipate excess excitation energy(e.g.,non-photochemical quenching)or reduction power(e.g.,flavodiiron proteins).Moreover,schemes for improving acclimation,inspired by natural or laboratory-induced adaptation,are introduced.However,all these endeavors are still in an early exploratory phase and/or have not resulted in the desired outcome,largely because photosynthesis is embedded within large networks of closely interacting cellular and metabolic processes,which can vary among species and even cultivars.This explains why integrated,systems-wide approaches are required to achieve the breakthroughs required for effectively increasing crop yields.

Theplant cytosolic m^(6)A RNA methylome stabilizes photosynthesis in the cold

The sessile lifestyle of plants requires an immediate response to environmental stressors that affect photosynthesis,growth,and crop yield.Here,we showed that three abiotic perturbations—heat,cold,and high light—triggered considerable changes in the expression signatures of 42 epitranscriptomic factors(writers,erasers,and readers)with putative chloroplast-associated functions that formed clusters of commonly expressed genes in Arabidopsis.The expression changes under all conditions were reversible upon deacclimation,identifying epitranscriptomic players as modulators in acclimation processes.Chloroplast dysfunctions,particularly those induced by the oxidative stress-inducing norflurazon in a largely GENOME UNCOUPLED-independent manner,triggered retrograde signals to remodel chloroplastassociated epitranscriptomic expression patterns.N6-methyladenosine(m^(6)A)is known as the most prevalent RNA modification and impacts numerous developmental and physiological functions in living organisms.During cold treatment,expression of components of the primary nuclear m^(6)A methyltransferase complex was upregulated,accompanied by a significant increase in cellular m^(6)A mRNA marks.In the cold,the presence of FIP37,a core component of the writer complex,played an important role in positive regulation of thylakoid structure,photosynthetic functions,and accumulation of photosystemⅠ,the Cytb6f complex,cyclic electron transport proteins,and Curvature Thylakoid1 but not that of photosystemⅡcomponents and the chloroplast ATP synthase.Downregulation of FIP37 affected abundance,polysomal loading,and translation of cytosolic transcripts related to photosynthesis in the cold,suggesting m^(6)Adependent translational regulation of chloroplast functions.In summary,we identified multifaceted roles of the cellular m^(6)A RNA methylome in coping with cold;these were predominantly associated with chloroplasts and served to stabilize photosynthesis.

COG1-A master transcription factor regulating photosynthesis

COG1 CANENHANCE PHOTOSYNTHESIS AND PLANT GROWTH Recently,Wei et al.(2023)report that overexpression or a gain-offunction mutation of COGWHEEL1(COG1)gene,which encodes a transcription factor belonging to the DNA binding with one finger(Dof)protein family,can increase growth and biomass in Arabidopsis thaliana.Similarly,overexpression of any of its six paralogs,CYCLING DOF FACTORS 1-6,also leads to increased growth and biomass production,implying that these seven Dof proteins have partially redundant functions.Accordingly,several of the seven Dof genes must be knocked out to significantly reduce growth and biomass production;the septuple mutants show severely reduced growth but are still viable.COG1 positively modulates the accumulation of transcripts of several photosynthetic genes and,in turn,the corresponding proteins and binds to the promoters of most LHC genes.Changes in gene expression related to photosynthesis increase the efficiency of photosynthesis.Overexpression of two closely related Brassica rapa COG1 homologs in A.thaliana or Arabidopsis COG1 in Nicotiana benthamiana,could cause an increase in growth and biomass production.These results indicate that the effects of COG1 are not confined to a specific species.

The Plastid Envelope CHLOROPLAST MANGANESE TRANSPORTER1 Is Essential for Manganese Homeostasis in Arabidopsis

The transition metal manganese （Mn） is indispensable for photoautotrophic growth since photosystem II （PSII） employs an inorganic Mn4CaOs cluster for water splitting. Here, we show that the Arabidopsis membrane protein CHLOROPLAST MANGANESE TRANSPORTER1 （CMT1） is involved in chloroplast Mn homeostasis. CMT1 is the closest homolog of the previously characterized thylakoid Mn transporter PHOTOSYNTHESIS-AFFECTED MUTANT71 （PAM71）. In contrast to PAM71, CMT1 resides at the chloro- plast envelope and is ubiquitously expressed. Nonetheless, like PAM71, the expression of CMT1 can also alleviate the Mn-sensitive phenotype of yeast mutant qomrl. The cmtl mutant is severely suppressed in growth, chloroplast ultrastructure, and PSII activity owing to a decrease in the amounts of pigments and thylakoid membrane proteins. The importance of CMT1 for chloroplast Mn homeostasis is demonstratedby the significant reduction in chloroplast Mn concentrations in cmtl-1, which exhibited reduced Mn binding in PSII complexes. Moreover, CMT1 expression is downregulated in Mn-surplus conditions. The pam71 cmtl-ldouble mutant resembles the cmtl-f single mutant rather than pare71 in most respects. Taken together, our results suggest that CMT1 mediates Mn2 uptake into the chloroplast stroma, and that CMT1 and PAM71 function sequentially in Mn delivery to PSII across the chloroplast envelope and the thylakoid membrane.

Update on Chloroplast Research： New Tools, New Topics, and New Trends

Chloroplasts, the green differentiation form of plastids, are the sites of photosynthesis and other important plant functions. Genetic and genomic technologies have greatly boosted the rate of discovery and functional character- ization of chloroplast proteins during the past decade. Indeed, data obtained using high-throughput methodologies, in particular proteomics and transcriptomics, are now routinely used to assign functions to chloroplast proteins. Our knowl- edge of many chloroplast processes, notably photosynthesis and photorespiration, has reached such an advanced state that biotechnological approaches to crop improvement now seem feasible. Meanwhile, efforts to identify the entire com- plement of chloroplast proteins and their interactions are progressing rapidly, making the organelle a prime target for systems biology research in plants.

SNOWY COTYLEDON 2 Promotes Chloroplast Development and Has a Role in Leaf Variegation in Both Lotus japonicus and Arabidopsis thaliana

Plants contain various factors that transiently interact with subunits or intermediates of the thylakoid multiprotein complexes, promoting their stable association and integration. Hence, assembly factors are essential for chloroplast development and the transition from heterotrophic to phototrophic growth. Snowy cotyledon 2 （SCO2） is a DNAJ-like protein involved in thylakoid membrane biogenesis and interacts with the light-harvesting chlorophyll-binding protein LHCBI. In Arabidopsis thaliana, SCO2 function was previ- ously reported to be restricted to cotyledons. Here we show that disruption of SC02 in Lotus japonicus results not only in paler cotyledons but also in variegated true leaves. Furthermore, smaller and pale- green true leaves can also be observed in A. thaliana sco2 （atsco2） mutants under short-day conditions. In both species, SCO2 is required for proper accumulation of PSlI-LHCll complexes. In contrast to other variegated mutants, inhibition of chloroplastic translation strongly affects L. japonicus sco2 mutant devel- opment and fails to suppress their variegated phenotype. Moreover, inactivation of the suppressor of variegation AtClpR1 in the atsco2 background results in an additive double-mutant phenotype with variegated true leaves. Taken together, our results indicate that SCO2 plays a distinct role in PSll assembly or repair and constitutes a novel factor involved in leaf variegation.

PGR5-PGRL1-Dependent Cyclic Electron Transport Modulates Linear Electron Transport Rate in Arabidopsis thaliana

Plants need tight regulation of photosynthetic electron transport for survival and growth under environ- mental and metabolic conditions. For this purpose, the linear electron transport （LET） pathway is supple- mented by a number of alternative electron transfer pathways and valves. In Arabidopsis, cyclic electron transport （CET） around photosystem I （PSI）, which recycles electrons from ferrodoxin to plastoquinone, is the most investigated alternative route. However, the interdependence of LET and CET and the relative importance of CET remain unclear, largely due to the difficulties in precise assessment of the contribution of CET in the presence of LET, which dominates electron flow under physiological conditions. We there- fore generated Arabidopsis mutants with a minimal water-splitting activity, and thus a low rate of LET, by combining knockout mutations in Psb01, PsbP2, PsbQ1, PsbQ2, and PsbR loci. The resulting 45 mutant is viable, although mature leaves contain only ~20% of wild-type naturally less abundant Psb02 protein. 45 plants compensate for the reduction in LET by increasing the rate of CET, and inducing a strong non-photochemical quenching （NPQ） response during dark-to-light transitions. To identify the molecular origin of such a high-capacity CET, we constructed three sextuple mutants lacking the qE component of NPQ （45 npq4-1）, NDH-mediated CET （45 crr4-3）, or PGR5-PGRLl-mediated CET （45 pgrS）. Their analysis revealed that PGR5-PGRLl-mediated CET plays a major role in ~pH formation and induction of NPQ in C3 plants. Moreover, while pgr5 dies at the seedling stage under fluctuating light conditions, 45 pgr5 plants are able to survive, which underlines the importance of PGR5 in modulating the intersystem electron transfer.

High non-photochemical quenching of VPZ transgenic potato plants limits CO_(2) assimilation under high light conditions and reduces tuber yield under fluctuating light

Under natural conditions,photosynthesis has to be adjusted to fluctuating light intensities.Leaves exposed to high light dissipate excess light energy in form of heat at photosystem II(PSII)by a process called non-photochemical quenching(NPQ).Upon fast transition from light to shade,plants lose light energy by a relatively slow relaxation from photoprotection.Combined overexpression of violaxanthin de-epoxidase(VDE),PSII subunit S(PsbS)and zeaxanthin epoxidase(ZEP)in tobacco accelerates relaxation from photoprotection,and increases photosynthetic productivity.In Arabidopsis,expression of the same three genes(VPZ)resulted in a more rapid photoprotection but growth of the transgenic plants was impaired.Here we report on VPZ expressing potato plants grown under various light regimes.Similar to tobacco and Arabidopsis,induction and relaxation of NPQ was accelerated under all growth conditions tested,but did not cause an overall increased photosynthetic rate or growth of transgenic plants.Tuber yield of VPZ expressing plants was unaltered as compared to control plants under constant light conditions and even decreased under fluctuating light conditions.Under control conditions,levels of the phytohormone abscisic acid(ABA)were found to be elevated,indicating an increased violaxanthin availability in VPZ plants.However,the increased basal ABA levels did not improve drought tolerance of VPZ transgenic potato plants under greenhouse conditions.The failure to benefit from improved photoprotection is most likely caused by a reduced radiation use efficiency under high light conditions resulting from a too strong NPQ induction.Mitigating this negative effect in the future might help to improve photosynthetic performance in VPZ expressing potato plants.

Meta-Analysis of Retrograde Signaling in Arabidopsis thaliana Reveals a Core Module of Genes Embedded in Complex Cellular Signaling Networks

Plastid-to-nucleus signaling is essential for the coordination and adjustment of cellular metabolism in response to environmental and developmental cues of plant cells. A variety of operational retrograde signaling path- ways have been described that are thought to be triggered by reactive oxygen species, photosynthesis redox imbalance, tetrapyrrole intermediates, and other metabolic traits. Here we report a meta-analysis based on transcriptome and pro- tein interaction data. Comparing the output of these pathways reveals the commonalities and peculiarities stimulated by six different sources impinging on operational retrograde signaling. Our study provides novel insights into the interplay of these pathways, supporting the existence of an as-yet unknown core response module of genes being regulated under all conditions tested. Our analysis further highlights affiliated regulatory cis-elements and classifies abscisic acid and auxin-based signaling as secondary components involved in the response cascades following a plastidial signal. Our study provides a global analysis of structure and interfaces of different pathways involved in plastid-to-nucleus signaling and a new view on this complex cellular communication network.

The Arabidopsis Protein CGLD11 Is Required for Chloroplast ATP Synthase Accumulation

ATP synthases in chloroplasts （cpATPase） and mitochondria （mtATPase） are responsible for ATP production during photosynthesis and oxidative phosphorylation, respectively. Both enzymes consist of two multi- subunit complexes, the membrane-bound coupling factor O and the soluble coupling factor 1. During cpATPase biosynthesis, several accessory factors facilitate subunit production and orchestrate complex assembly. Here, we describe a new auxiliary protein in Arabidopsis thaliana, which is required for cpATPase accumulation. AtCGLD11 （CONSERVED IN THE GREEN LINEAGE AND DIATOMS 11） is a protein without any known functional domain and shows dual localization to chloroplasts and mitochondria. Loss of AtCGLDll function results in reduced levels of cpATPase and impaired photosynthetic performance with lower rates of ATP synthesis. In yeast two-hybrid experiments, AtCGLD11 interacts with the 13 subunits of the cpATPase and mtATPase. Our results suggest that AtCGLD11 functions in F1 assembly during cpATPase biogenesis, while its role in mtATPase biosynthesis may not, or not yet, be essential.

Extending the Repertoire of mTERF Proteins with Functions in Organellar Gene Expression

In plants,genetic information is stored in three different locations:the nucleus,the plastids,and the mitochondria.The genomes of the latter organelles code for only a tiny fraction of their respective proteomes,but they nevertheless require fully functional gene-expression machineries,which are far more complex than those of their prokaryotic progenitors.This is because the organellar gene expression(OGE)apparatus is a hybrid system,which combines features of the prokaryotic gene-expression apparatus with eukaryotic inventions(Pfannschmidt et al.,2015).In the plastids of higher plants,transcription is performed by three different RNA polymerases:two monomeric,nucleus-encoded RNA polymerases and a plastid-encoded(PEP)Escherichia coli-like enzyme(Pfannschmidt et al.,2015).

Mutants, Overexpressors, and Interactors of Arabidopsis Plastocyanin Isoforms： Revised Roles of Plastocyanin in Photosynthetic Electron Flow and Thylakoid Redox State

Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis thaliana. The PETE2 transcript is expressed at considerably higher levels and the PETE2 protein is the more abundant isoform. Null mutations in the PETE genes resulted in plants, designated pete1 and pete2, with decreased plastocyanin contents. However, despite reducing plastocyanin levels by over -90%, a pete2 null mutation on its own affects rates of photosynthesis and growth only slightly, whereas pete1 knockout plants, with about 60-80% of the wild-type plastocyanin level, did not show any alteration. Hence, plastocyanin concentration is not limiting for photosynthetic elec- tron flow under optimal growth conditions, perhaps implying other possible physiological roles for the protein. Indeed, plastocyanin has been proposed previously to cooperate with cytochrome C6A （Cyt C6A） in thylakoid redox reactions, but we find no evidence for a physical interaction between the two proteins, using interaction assays in yeast. We observed homodimerization of Cyt C6A in yeast interaction assays, but also Cyt C6A homodimers failed to interact with plastocyanin. Moreover, phenotypic analysis of atc6-1 pete1 and atc6-1 pete2 double mutants, each lacking Cyt C6A and one of the two piastocyanin-encoding genes, failed to reveal any genetic interaction. Overexpression of either PETE1 or PETE2 in the pete1 pete2 double knockout mutant background results in essentially wild-type photosynthetic performance, excluding the possibility that the two plastocyanin isoforms could have distinct functions in thylakoid electron flow.

Intracellular Communication

Gene expression of the nuclear, plastid, and mitochon- drial genomes in plants is mutually dependent and highly coordinately regulated. This ensures adequate synthesis of proteins functioning in common protein complexes for organellar gene expression and energy-transducing activities in chloroplasts and mitochondria. The concept of intracellular communication includes an anterograde and retrograde signaling network between nucleus and the two organelles, enabling this interactive exchange of information essential for cellular hemostasis. By definition, the intracellular communication is based on the inter-orga- nellar reciprocity whereby the transcriptional activities in the nucleus are regulated in part by signaling pathways derived from plastids and mitochondria （retrograde sign- aling）, while the organellar gene expression is controlled by information received form the nucleus （anterograde signaling）.