Conservation and specialization of the Ycf2-FtsHi chloroplast protein import motor in green algae.

The protein import motor in chloroplasts plays a pivotal role in their biogenesis and homeostasis by driving the translocation of preproteins into chloroplasts. While the Ycf2-FtsHi complex serves as the import motor in land plants, its evolutionary conservation, specialization, and mechanisms across photosynthetic organisms are largely unexplored. Here, we isolated and determined the cryogenic electron microscopy (cryo-EM) structures of the native Ycf2-FtsHi complex from Chlamydomonas reinhardtii, uncovering a complex composed of up to 19 subunits, including multiple green-algae-specific components. The heterohexameric AAA+ ATPase motor module is tilted, potentially facilitating preprotein handover from the translocon at the inner chloroplast membrane (TIC) complex. Preprotein interacts with Ycf2-FtsHi and enhances its ATPase activity in vitro. Integrating Ycf2-FtsHi and translocon at the outer chloroplast membrane (TOC)-TIC supercomplex structures reveals insights into their physical and functional interplay during preprotein translocation. By comparing these findings with those from land plants, our study establishes a structural foundation for understanding the assembly, function, evolutionary conservation, and diversity of chloroplast protein import motors.

STIC2 selectively binds ribosome-nascent chain complexes in the cotranslational sorting of Arabidopsis thylakoid proteins.

Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled ribosome-nascent chain complexes (RNCs) bearing D1 nascent chains. Stalled RNCs translating the soluble ribosomal subunit uS2c were used for comparison. Quantitative tandem-mass spectrometry of the purified RNCs identified around 140 proteins specifically associated with D1 RNCs, mainly involved in protein and cofactor biogenesis, including chlorophyll biosynthesis, and other metabolic pathways. Functional analysis of STIC2, a newly identified D1 RNC interactor, revealed its cooperation with chloroplast protein SRP54 in the de novo biogenesis and repair of D1, and potentially other cotranslationally-targeted reaction center subunits of PSII and PSI. The primary binding interface between STIC2 and the thylakoid insertase Alb3 and its homolog Alb4 was mapped to STIC2's ß-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4.

Selective autophagy regulates chloroplast protein import and promotes plant stress tolerance.

Chloroplasts are plant organelles responsible for photosynthesis and environmental sensing. Most chloroplast proteins are imported from the cytosol through the translocon at the outer envelope membrane of chloroplasts (TOC). Previous work has shown that TOC components are regulated by the ubiquitin-proteasome system (UPS) to control the chloroplast proteome, which is crucial for the organelle's function and plant development. Here, we demonstrate that the TOC apparatus is also subject to K63-linked polyubiquitination and regulation by selective autophagy, potentially promoting plant stress tolerance. We identify NBR1 as a selective autophagy adaptor targeting TOC components, and mediating their relocation into vacuoles for autophagic degradation. Such selective autophagy is shown to control TOC protein levels and chloroplast protein import and to influence photosynthetic activity as well as tolerance to UV-B irradiation and heat stress in Arabidopsis plants. These findings uncover the vital role of selective autophagy in the proteolytic regulation of specific chloroplast proteins, and how dynamic control of chloroplast protein import is critically important for plants to cope with challenging environments.

CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

Intra-chloroplast proteases: A holistic network view of chloroplast proteolysis.

Different proteases and peptidases are present within chloroplasts and nonphotosynthetic plastids to process precursor proteins and to degrade cleaved chloroplast transit peptides and damaged, misfolded, or otherwise unwanted proteins. Collectively, these proteases and peptidases form a proteolysis network, with complementary activities and hierarchies, and build-in redundancies. Furthermore, this network is distributed across the different intra-chloroplast compartments (lumen, thylakoid, stroma, envelope). The challenge is to determine the contributions of each peptidase (system) to this network in chloroplasts and nonphotosynthetic plastids. This will require an understanding of substrate recognition mechanisms, degrons, substrate, and product size limitations, as well as the capacity and degradation kinetics of each protease. Multiple extra-plastidial degradation pathways complement these intra-chloroplast proteases. This review summarizes our current understanding of these intra-chloroplast proteases in Arabidopsis and crop plants with an emphasis on considerations for building a qualitative and quantitative network view.

A cargo sorting receptor mediates chloroplast protein trafficking through the secretory pathway.

Nucleus-encoded chloroplast proteins can be transported via the secretory pathway. The molecular mechanisms underlying the trafficking of chloroplast proteins between the intracellular compartments are largely unclear, and a cargo sorting receptor has not previously been identified in the secretory pathway. Here, we report a cargo sorting receptor that is specifically present in Viridiplantae and mediates the transport of cargo proteins to the chloroplast. Using a forward genetic analysis, we identified a gene encoding a transmembrane protein (MtTP930) in barrel medic (Medicago truncatula). Mutation of MtTP930 resulted in impaired chloroplast function and a dwarf phenotype. MtTP930 is highly expressed in the aerial parts of the plant and is localized to the endoplasmic reticulum (ER) exit sites and Golgi. MtTP930 contains typical cargo sorting receptor motifs, interacts with Sar1, Sec12, and Sec24, and participates in coat protein complex II vesicular transport. Importantly, MtTP930 can recognize the cargo proteins plastidial N-glycosylated nucleotide pyrophosphatase/phosphodiesterase (MtNPP) and α-carbonic anhydrase (MtCAH) in the ER and then transport them to the chloroplast via the secretory pathway. Mutation of a homolog of MtTP930 in Arabidopsis (Arabidopsis thaliana) resulted in a similar dwarf phenotype. Furthermore, MtNPP-GFP failed to localize to chloroplasts when transgenically expressed in Attp930 protoplasts, implying that these cargo sorting receptors are conserved in plants. These findings fill a gap in our understanding of the mechanism by which chloroplast proteins are sorted and transported via the secretory pathway.

Identification of a highly efficient chloroplast-targeting peptide for plastid engineering.

Plastids are pivotal target organelles for comprehensively enhancing photosynthetic and metabolic traits in plants via plastid engineering. Plastidial proteins predominantly originate in the nucleus and must traverse membrane-bound multiprotein translocons to access these organelles. This import process is meticulously regulated by chloroplast-targeting peptides (cTPs). Whereas many cTPs have been employed to guide recombinantly expressed functional proteins to chloroplasts, there is a critical need for more efficient cTPs. Here, we performed a comprehensive exploration and comparative assessment of an advanced suite of cTPs exhibiting superior targeting capabilities. We employed a multifaceted approach encompassing computational prediction, in planta expression, fluorescence tracking, and in vitro chloroplast import studies to identify and analyze 88 cTPs associated with Arabidopsis thaliana mutants with phenotypes linked to chloroplast function. These polypeptides exhibited distinct abilities to transport green fluorescent protein (GFP) to various compartments within leaf cells, particularly chloroplasts. A highly efficient cTP derived from Arabidopsis plastid ribosomal protein L35 (At2g24090) displayed remarkable effectiveness in chloroplast localization. This cTP facilitated the activities of chloroplast-targeted RNA-processing proteins and metabolic enzymes within plastids. This cTP could serve as an ideal transit peptide for precisely targeting biomolecules to plastids, leading to advancements in plastid engineering.

THYLAKOID FORMATION 1 interacts with FLOWERING LOCUS T and modulates temperature-responsive flowering in Arabidopsis.

The intracellular localization of the florigen FLOWERING LOCUS T (FT) is important for its long-distance transport toward the shoot apical meristem. However, the mechanisms regulating the FT localization remain poorly understood. Here, we discovered that in Arabidopsis thaliana, the chloroplast-localized protein THYLAKOID FORMATION 1 (THF1) physically interacts with FT, sequestering FT in the outer chloroplast envelope. Loss of THF1 function led to temperature-insensitive flowering, resulting in early flowering, especially under low ambient temperatures. THF1 mainly acts in the leaf vasculature and shoot apex to prevent flowering. Mutation of CONSTANS or FT completely suppressed the early flowering of thf1-1 mutants. FT and THF1 interact via their anion binding pocket and coiled-coil domain (CCD), respectively. Deletion of the CCD in THF1 by gene editing caused temperature-insensitive early flowering similar to that observed in the thf1-1 mutant. FT levels in the outer chloroplast envelope decreased in the thf1-1 mutant, suggesting that THF1 is important for sequestering FT. Furthermore, THF1 protein levels decreased in seedlings grown at high ambient temperature, suggesting an explanation for its role in plant responses to ambient temperature. A thf1-1 phosphatidylglycerolphosphate synthase 1 (pgp1) double mutant exhibited additive acceleration of flowering at 23 and 16°C, compared to the single mutants, indicating that THF1 and phosphatidylglycerol (PG) act as independent but synergistic regulators of temperature-responsive flowering. Collectively, our results provide an understanding of the genetic pathway involving THF1 and its role in temperature-responsive flowering and reveal a previously unappreciated additive interplay between THF1 and PG in temperature-responsive flowering.

Chloroplast protein translocation pathways and ubiquitin-dependent regulation at a glance.

Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the ∼3000 proteins in chloroplasts are nucleus encoded and must be imported from the cytosol. Thus, the protein import machinery of the organelle (the TOC-TIC apparatus) is of fundamental importance for chloroplast biogenesis and operation. Cytosolic factors target chloroplast precursor proteins to the TOC-TIC apparatus, which drives protein import across the envelope membranes into the organelle, before various internal systems mediate downstream routing to different suborganellar compartments. The protein import system is proteolytically regulated by the ubiquitin-proteasome system (UPS), enabling centralized control over the organellar proteome. In addition, the UPS targets a range of chloroplast proteins directly. In this Cell Science at a Glance article and the accompanying poster, we present mechanistic details of these different chloroplast protein targeting and translocation events, and of the UPS systems that regulate chloroplast proteins.

Chloroplast biogenesis involves spatial coordination of nuclear and organellar gene expression in Chlamydomonas.

The localization of translation can direct the polypeptide product to the proper intracellular compartment. Our results reveal translation by cytosolic ribosomes on a domain of the chloroplast envelope in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii). We show that this envelope domain of isolated chloroplasts retains translationally active ribosomes and mRNAs encoding chloroplast proteins. This domain is aligned with localized translation by chloroplast ribosomes in the translation zone, a chloroplast compartment where photosystem subunits encoded by the plastid genome are synthesized and assembled. Roles of localized translation in directing newly synthesized subunits of photosynthesis complexes to discrete regions within the chloroplast for their assembly are suggested by differences in localization on the chloroplast of mRNAs encoding either subunit of the light-harvesting complex II or the small subunit of Rubisco. Transcription of the chloroplast genome is spatially coordinated with translation, as revealed by our demonstration of a subpopulation of transcriptionally active chloroplast nucleoids at the translation zone. We propose that the expression of chloroplast proteins by the nuclear-cytosolic and organellar genetic systems is organized in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes.

REDUCED CHLOROPLAST COVERAGE proteins are required for plastid proliferation and carotenoid accumulation in tomato.

Increasing the amount of cellular space allocated to plastids will lead to increases in the quality and yield of crop plants. However, mechanisms that allocate cellular space to plastids remain poorly understood. To test whether the tomato (Solanum lycopersicum L.) REDUCED CHLOROPLAST COVERAGE (SlREC) gene products serve as central components of the mechanism that allocates cellular space to plastids and contribute to the quality of tomato fruit, we knocked out the 4-member SlREC gene family. We found that slrec mutants accumulated lower levels of chlorophyll in leaves and fruits, accumulated lower levels of carotenoids in flowers and fruits, allocated less cellular space to plastids in leaf mesophyll and fruit pericarp cells, and developed abnormal plastids in flowers and fruits. Fruits produced by slrec mutants initiated ripening later than wild type and produced abnormal levels of ethylene and abscisic acid (ABA). Metabolome and transcriptome analyses of slrec mutant fruits indicated that the SlREC gene products markedly influence plastid-related gene expression, primary and specialized metabolism, and the response to biotic stress. Our findings and previous work with distinct species indicate that REC proteins help allocate cellular space to plastids in diverse species and cell types and, thus, play a central role in allocating cellular space to plastids. Moreover, the SlREC proteins are required for the high-level accumulation of chlorophyll and carotenoids in diverse organs, including fruits, promote the development of plastids and influence fruit ripening by acting both upstream and downstream of ABA biosynthesis in a complex network.

Mutations in the chloroplast inner envelope protein TIC100 impair and repair chloroplast protein import and impact retrograde signaling.

Chloroplast biogenesis requires synthesis of proteins in the nucleocytoplasm and the chloroplast itself. Nucleus-encoded chloroplast proteins are imported via multiprotein translocons in the organelle's envelope membranes. Controversy exists around whether a 1-MDa complex comprising TIC20, TIC100, and other proteins constitutes the inner membrane TIC translocon. The Arabidopsis thaliana cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100. The tic100cue8 mutant accumulates reduced levels of 1-MDa complex components and exhibits reduced import of two nucleus-encoded chloroplast proteins of different import profiles. A search for suppressors of tic100cue8 identified a second mutation within the same gene, tic100soh1, which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100soh1 retains but rapidly exits virescence and rescues the synthetic lethality of tic100cue8 when retrograde signaling is impaired by a mutation in the GENOMES UNCOUPLED 1 gene. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signaling response is triggered when TIC100 function is altered. Our results are consistent with a role for TIC100, and by extension the 1-MDa complex, in the chloroplast import of photosynthetic and nonphotosynthetic proteins, a process which initiates retrograde signaling.

TIC236 gain-of-function mutations unveil the link between plastid division and plastid protein import.

SignificanceAlthough 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.

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

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

Divergent Protein Redox Dynamics and Their Relationship with Electron Transport Efficiency during Photosynthesis Induction.

Various chloroplast proteins are activated/deactivated during the light/dark cycle via the redox regulation system. Although the photosynthetic electron transport chain provides reducing power to redox-sensitive proteins via the ferredoxin (Fd)/thioredoxin (Trx) pathway for their enzymatic activity control, how the redox states of individual proteins are linked to electron transport efficiency remains uncharacterized. Here we addressed this subject with a focus on the photosynthetic induction phase. We used Arabidopsis plants, in which the amount of Fd-Trx reductase (FTR), a core component in the Fd/Trx pathway, was genetically altered. Several chloroplast proteins showed different redox shift responses toward low- and high-light treatments. The light-dependent reduction of Calvin-Benson cycle enzymes fructose 1,6-bisphosphatase (FBPase) and sedoheptulose 1,7-bisphosphatase (SBPase) was partially impaired in the FTR-knockdown ftrb mutant. Simultaneous analyses of chlorophyll fluorescence and P700 absorbance change indicated that the induction of the electron transport reactions was delayed in the ftrb mutant. FTR overexpression also mildly affected the reduction patterns of FBPase and SBPase under high-light conditions, which were accompanied by the modification of electron transport properties. Accordingly, the redox states of FBPase and SBPase were linearly correlated with electron transport rates. In contrast, ATP synthase was highly reduced even when electron transport reactions were not fully induced. Furthermore, the redox response of proton gradient regulation 5-like photosynthetic phenotype1 (PGRL1; a protein involved in cyclic electron transport) did not correlate with electron transport rates. Our results provide insights into the working dynamics of the redox regulation system and their differential associations with photosynthetic electron transport efficiency.

Mutant noxy8 exposes functional specificities between the chloroplast chaperones CLPC1 and CLPC2 in the response to organelle stress and plant defence.

Chloroplast function is essential for growth, development, and plant adaptation to stress. Organelle stress and plant defence responses were examined here using noxy8 (nonresponding to oxylipins 8) from a series of Arabidopsis mutants. The noxy8 mutation was located at the CLPC2 gene, encoding a chloroplast chaperone of the protease complex CLP. Although its CLPC1 paralogue is considered to generate redundancy, our data reveal significant differences distinguishing CLPC2 and CLPC1 functions. As such, clpc1 mutants displayed a major defect in housekeeping chloroplast proteostasis, leading to a pronounced reduction in growth and pigment levels, enhanced accumulation of chloroplast and cytosol chaperones, and resistance to fosmidomycin. Conversely, clpc2 mutants showed severe susceptibility to lincomycin inhibition of chloroplast translation and resistance to Antimycin A inhibition of mitochondrial respiration. In the response to Pseudomonas syringae pv. tomato, clpc2 but not clpc1 mutants were resistant to bacterial infection, showing higher salicylic acid levels, defence gene expression and 9-LOX pathway activation. Our findings suggest CLPC2 and CLPC1 functional specificity, with a preferential involvement of CLPC1 in housekeeping processes and of CLPC2 in stress responses.

The role of chloroplast SRP54 domains and its C-terminal tail region in post- and co-translational protein transport in vivo.

In the chloroplast, the 54 kDa subunit of the signal recognition particle (cpSRP54) is involved in the post-translational transport of the light-harvesting chlorophyll a/b-binding proteins (LHCPs) and the co-translational transport of plastid-encoded subunits of the photosynthetic complexes to the thylakoid membrane. It forms a high-affinity complex with plastid-specific cpSRP43 for post-translational transport, while a ribosome-associated pool coordinates its co-translational function. CpSRP54 constitutes a conserved multidomain protein, comprising a GTPase (NG) and a methionine-rich (M) domain linked by a flexible region. It is further characterized by a plastid-specific C-terminal tail region containing the cpSRP43-binding motif. To characterize the physiological role of the various regions of cpSRP54 in thylakoid membrane protein transport, we generated Arabidopsis cpSRP54 knockout (ffc1-2) lines producing truncated cpSRP54 variants or a GTPase point mutation variant. Phenotypic characterization of the complementation lines demonstrated that the C-terminal tail region of cpSRP54 plays an important role exclusively in post-translational LHCP transport. Furthermore, we show that the GTPase activity of cpSRP54 plays an essential role in the transport pathways for both nuclear as well as plastid-encoded proteins. In addition, our data revealed that plants expressing cpSRP54 without the C-terminal region exhibit a strongly increased accumulation of a photosystem I assembly intermediate.

Genome-wide signatures of plastid-nuclear coevolution point to repeated perturbations of plastid proteostasis systems across angiosperms.

Nuclear and plastid (chloroplast) genomes experience different mutation rates, levels of selection, and transmission modes, yet key cellular functions depend on their coordinated interactions. Functionally related proteins often show correlated changes in rates of sequence evolution across a phylogeny [evolutionary rate covariation (ERC)], offering a means to detect previously unidentified suites of coevolving and cofunctional genes. We performed phylogenomic analyses across angiosperm diversity, scanning the nuclear genome for genes that exhibit ERC with plastid genes. As expected, the strongest hits were highly enriched for genes encoding plastid-targeted proteins, providing evidence that cytonuclear interactions affect rates of molecular evolution at genome-wide scales. Many identified nuclear genes functioned in post-transcriptional regulation and the maintenance of protein homeostasis (proteostasis), including protein translation (in both the plastid and cytosol), import, quality control, and turnover. We also identified nuclear genes that exhibit strong signatures of coevolution with the plastid genome, but their encoded proteins lack organellar-targeting annotations, making them candidates for having previously undescribed roles in plastids. In sum, our genome-wide analyses reveal that plastid-nuclear coevolution extends beyond the intimate molecular interactions within chloroplast enzyme complexes and may be driven by frequent rewiring of the machinery responsible for maintenance of plastid proteostasis in angiosperms.

TIC236 links the outer and inner membrane translocons of the chloroplast.

The two-membrane envelope is a defining feature of chloroplasts. Chloroplasts evolved from a Gram-negative cyanobacterial endosymbiont. During evolution, genes of the endosymbiont have been transferred to the host nuclear genome. Most chloroplast proteins are synthesized in the cytosol as higher-molecular-mass preproteins with an N-terminal transit peptide. Preproteins are transported into chloroplasts by the TOC and TIC (translocons at the outer- and inner-envelope membranes of chloroplasts, respectively) machineries1,2, but how TOC and TIC are assembled together is unknown. Here we report the identification of the TIC component TIC236; TIC236 is an integral inner-membrane protein that projects a 230-kDa domain into the intermembrane space, which binds directly to the outer-membrane channel TOC75. The knockout mutation of TIC236 is embryonically lethal. In TIC236-knockdown mutants, a smaller amount of the inner-membrane channel TIC20 was associated with TOC75; the amount of TOC-TIC supercomplexes was also reduced. This resulted in a reduced import rate into the stroma, though outer-membrane protein insertion was unaffected. The size and the essential nature of TIC236 indicate that-unlike in mitochondria, in which the outer- and inner-membrane translocons exist as separate complexes and a supercomplex is only transiently assembled during preprotein translocation3,4-a long and stable protein bridge in the intermembrane space is required for protein translocation into chloroplasts. Furthermore, TIC236 and TOC75 are homologues of bacterial inner-membrane TamB5 and outer-membrane BamA, respectively. Our evolutionary analyses show that, similar to TOC75, TIC236 is preserved only in plants and has co-evolved with TOC75 throughout the plant lineage. This suggests that the backbone of the chloroplast protein-import machinery evolved from the bacterial TamB-BamA protein-secretion system.

Immunofluorescence staining of chloroplast proteins with frozen sections of plant tissues.

KEY MESSAGE: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.