GET3B is involved in chloroplast biogenesis and interacts with the thylakoidal ALB3 and ALB4 insertases

GET3B is involved in chloroplast biogenesis and interacts with the thylakoidal ALB3 and ALB4 insertases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article GET3B is involved in chloroplast biogenesis and interacts with the thylakoidal ALB3 and ALB4 insertases Uwe Sakamuzi Bodensohn, Beatrix Dünschede, Chiara Kuhlmann, Khushbu Kumari, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6212458/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Apr, 2025 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract Protein targeting and insertion into membranes are essential for cellular organization and organelle function. The Guided Entry of Tail-anchored (GET) pathway facilitates the post-translational targeting and insertion of tail-anchored (TA) membrane proteins. Arabidopsis thaliana has four GET3 homologues, including At GET3B and At GET3D localized to chloroplasts. These photosynthetic organelles possess complex membrane systems, and the mechanisms underlying their protein targeting and membrane biogenesis are not fully understood. This study conducted a comprehensive proteomic analysis of get3b mutant plastids, which displayed significant alterations. Fluorometric based complex assembly as well as CO 2 assimilation analyses confirmed that disruption of GET3B function displayed a significant impact on photosystem II assembly as well as carbon fixation, respectively, indicating a functional role in chloroplast biogenesis. Additionally, genetic interactions were found between GET3B and the two component STIC system, which cooperates with the cpSRP pathway and is involved in the co-translational sorting of thylakoid proteins. Further, physical interactions were observed between GET3B and the C-terminus of ALB3 and ALB4 in vitro and the full length proteins in vivo , indicating a role of GET3B in protein targeting and membrane integration within chloroplasts. These findings enhance our understanding of GET3B’s involvement in stromal protein targeting and thylakoidal biogenesis. ALB3 ALB4 cpSRP54 GET3B STIC1 STIC2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key message Proteomic, functional physiological analyses of get3b mutant plants highlight GET3B’s role in chloroplast function. Genetic and interaction analyses indicate get3b and srp54 as mutual potentiators that might share terminal insertases. Introduction Intrinsic membrane proteins (IMPs) represent an extremely diversified group that represents roughly 30% of the cellular proteome (Wallin and Von Heijne, 1998 ). Tail anchored (TA) proteins share a simple type II topology with a cytosolically exposed N-terminus and a single C-terminal transmembrane domain (TMD) within the last 50 amino acids (AAs) of the polypeptide stretch. Even though they constitute 3–5% of eukaryotic IMPs (Kalbfleisch et al., 2007 ), TA proteins fulfill a multitude of essential biological functions like apoptosis (Jiang et al., 2014 ), vesicle transport (van Berkel et al., 2020 ) and organelle biogenesis (Sommer et al., 2013 ). The guided entry of TA proteins (GET) pathway facilitates the post-translational targeting of this subset of IMPs. After translation is terminated, the pretargeting complex captures the TA protein and hands it over to the central ATPase of the GET pathway, Get3 (Wang et al., 2010 ). Thereafter the TA protein is passed on to its cognate receptor, which functions as an insertase (Wang et al., 2014 ). For reasons of simplicity, we will generally refer to “Get” factors and plant specific elements will be denoted in all capital letters, in agreement with Arabidopsis thaliana nomenclature. A characteristic trait of Get3 is the formation of a symmetric homodimer equipped with a static ATPase domain and a flexible α-helical domain that is sensitive to nucleotide binding. This nucleotide binding state induces the formation of a hydrophobic groove or chamber capable of binding TA substrate (Mateja et al., 2009; Bozkurt et al., 2009 , Mateja et., 2015, Barlow et al., 2023 ). In a cellular context, Get3 also has a secondary function in eukaryotes by acting as an ATP-independent holdase under energy limiting conditions and oxidative stress (Powis et al., 2013 ; Voth et al., 2014 ). Get3 and its orthologues TRC40/ArsA are highly conserved in all domains of life and have diversified into three clades, namely clade a, clade bc and clade d (Xing et al., 2017 ; Barlow et al., 2023 ). In contrast to metazoans and fungi, which solely contain clade a Get3 homologues, photosynthetic organisms contain multiple Get3 homologues from all clades (Xing et al., 2017 ; Bodensohn et al., 2019 ; Barlow et al., 2023 ). The three clades differ in their amino acid (AA) composition, which results in different domain architectures. In the majority of the cases clade a proteins have a cytosolic and clade bc an organellar localization (Xing et al., 2017 ; Bodensohn et al., 2019 ). Depending on the prokaryotic or eukaryotic origin of a photosynthetic organism, clade d proteins have a cytosolic or organellar localization, respectively (Barlow et al., 2023 ). Arabidopsis thaliana has four Get3 homologues, namely At GET3A, At GET3B, At GET3C and At GET3D localized to cytosol, chloroplasts, mitochondria and chloroplasts, respectively (Duncan et al., 2013 ; Xing et al., 2017 ; Bodensohn et al., 2019 ; Barlow et al., 2023 ). Loss of function mutations in any of the clade a or clade bc orthologues renders the resulting plant largely indistinguishable from the wild-type (Xing et al., 2017 ). This is also the case in yeast and nematodes in which the deletion of Get3 leads to conditional growth defects (Schuldiner et al., 2008 ; Tseng et al., 2007 ). In mammals however, the disruption of TRC40 is embryo lethal (Mukhopadhyay et al., 2006 ). In photosynthetic eukaryotes, plastids constitute one of the most complex systems in terms of protein sorting. 95% of their proteome is encoded by nuclear genes and precursor proteins are synthesized on cytosolic ribosomes to be imported post-translationally. This is facilitated by two multimeric translocons embedded in the outer (TOC) and inner (TIC) envelope that form a continuum from the cytosol, through the intermembrane space (IMS) into the stroma (Liu et al., 2023 ). Once arrived in the stroma, precursor proteins can be directed to numerous translocation machineries imbedded within the inner envelope or thylakoid membrane. This leads to six different sub organellar localizations that each can individually be accessed with different sets of protein targeting networks (Jarvis and López-Juez, 2013 ). Taking the pivotal role of plastids into account, the targeting and translocation networks must operate with high fidelity to ensure proper organellar biogenesis, repair and functionality. This is reflected by the deficiencies in plant fitness that arise when components of these are disrupted (Bauer et al., 2000 ; Amin et al., 1999 ; Pilgrim et al., 1998 ; Sundberg et al., 1997 ). One intensively studied stromal targeting pathway is the post-translational cpSRP pathway, which targets a single population of IMPs, namely light harvesting chlorophyll binding proteins, to the thylakoid membrane (Schuenemann et al., 1998 ; Ziehe et al., 2018 ). CpSRP is composed of cpSRP54 (henceforth referred to as SRP54), the plastidic homologue of a highly conserved subunit of the cytosolic SRP, and a plant specific targeting factor, cpSRP43 (SRP43 from here on). SRP54 can also interact with the plastidic 70S ribosome and mediates co-translational transport of substrate proteins to its respective insertase, ALB3 and the SecYE translocon (Hristou et al., 2019 ; Moore et al., 2003 ). The terminal insertase ALB3 belongs to the Oxa1 superfamily of membrane protein biogenesis factors which also includes the ER resident Get1 (Anghel et al., 2017 ). In plants, an additional homologue of ALB3 exists, ALB4 (Gerdes et al., 2006 ). ALB4 is involved in the assembly and stabilization of ATP synthase intermediates (Benz et al., 2009 ). Furthermore, subsequent analyses displayed that ALB4 works at least partially together with ALB3 and participates in the biogenesis of a subset of thylakoid membrane proteins. (Trösch et al., 2015 ). ALB4 also interacts with STIC2, a protein identified in a genetic screen for the suppressors of chlorotic tic40 , an A. thaliana mutant lacking the chloroplast inner envelope protein TIC40. The STIC system is described as a two component network composed of STIC1 (ALB4) and STIC2, which acts together in a shared pathway with cpSRP54 and cpFtsY (Bédard et al., 2017 ). STIC2 shares homology with the bacterial YbaB,(Link et al., 2008 ). Recently, STIC2, was identified as a factor associated with plastidic ribosomes translating the photosystem (PS) II reaction center protein D1, is likely involved in co-translational sorting of other plastid-encoded multi-pass IMPs (Stolle et al., 2024 ). In a previous study, we provided evidence that the At GET3B can selectively bind SECE1 and shows a genetic interaction with components of the cpSRP system. Furthermore, mutant plants showed altered levels of photosynthesis-related proteins (Anderson et al., 2021 ). This prompted us to investigate if GET3B interacts with constituents of the cpSRP and STIC systems, with an emphasis on their cognate receptors ALB3 and ALB4, respectively. We hypothesized that GET3B might play a crucial role in the targeting and membrane integration of specific proteins within chloroplasts, thereby contributing to the biogenesis and functionality of chloroplast membranes. To address this hypothesis, we employed a combination of biochemical, molecular, fluorometric and genetic approaches to provide evidence that GET3B physically interacts with the C-terminus of ALB3 as well as ALB4 and genetically interacts with components of the STIC and cpSRP pathways, and that its disruption has an effect on chloroplast biogenesis and function. Materials and Methods Plant materials and plant growth Arabidopsis thaliana seeds were surface sterilized and sown on 0.8% (w/v) agar plates containing ½ strength Murashige and Skoog media (Caisson Labs), 50mM MES-KOH, pH 5.7, and 1% (w/v) sucrose. Plants used for fluorometric measurements were germinated and cultivated on MS plates without sucrose. Following 2–3 days of stratification at 4°C, the plates were exposed to 100 µE m − 2 s − 1 of light provided by cool white (4100K) fluorescent bulbs on a 12 h light/12 h dark cycle for 7–14 days at 22°C, at which point seedlings were transplanted to a soil-less mix (Fafard Germinating Mix, Sungro Horticulture), watered with 10% Hoagland’s solution (Hoagland and Arnon, 1950 ), and grown under the same conditions as the plates. Columbia-0 ecotype was used as wild-type for all pictures and measurements in this study. Seeds for the T-DNA insertional mutants were acquired from the Arabidopsis Biological Resource Center ( https://abrc.osu.edu/ ). Mutant alleles previously characterized as resulting in loss of function were used for the genetic crosses: get3b-2 ( SALK_017702C, Xing et al., 2017 ), cpsrp54-3 (WiscDsLox289_292B14, Yu et al., 2012 ), alb4-1 (SALK_136199, Bédard et al., 2017 ), stic2-3 (SALK_001500, Bédard et al. 2017 ) and stic2-4 (WiscDsLox445D01, Bédard et al. 2017 ). The two stic2 alleles behaved similarly; therefore, we refer to stic2 mutants without the allele number in other sections of the paper. Lines carrying the mutant alleles were identified by PCR genotyping using the genotyping primers listed in Supplemental Table S2. Generation of transgenic plants Generation of the constructs used for moderate overexpression and genetic complementation experiments was previously described (Anderson et al. 2021 ). The ProUBI10:GET3B-STREP construct contains the promoter for the UBIQUITIN 10 gene (Norris et al., 1993 ), genomic sequence for the coding region of GET3B , sequence encoding a C-terminal 1X STREP tag, and the terminator of the NOS ( nopaline synthase ) gene. For the ProUBI10:GET3B(D124N)-STREP construct, the D124N mutation was introduced into the ProUBI10:GET3B-STREP construct by site-directed mutagenesis. The Arabidopsis plants transformed for the genetic complementation experiments were homozygous for the alb4 and get3b alleles, and heterozygous for the srp54 allele. Plants transformed for moderate overexpression studies were homozygous for either the alb4 or stic2-4 allele. The plant transformation procedure was previously described (Anderson et al. 2021 ), and transformants were selected in the T1 generation based on gentamycin resistance. Transformants containing mutant alleles as well as the introduced transgene were identified by PCR genotyping using primers listed in Supplemental Table S2. Genetic complementation was assessed in the T2 generation based on the phenotype of plants that were homozygous for alb4 , get3b and srp54 and carried the introduced transgene. The effects of moderate overexpression in alb4 or stic2 homozygous backgrounds were assessed in either the T2 or T3 generations. Rosette measurements For measurements of rosette diameters and chlorophyll content, plants with specific genotypes were grown for approximately 4 weeks under the conditions described above. Rosette diameter was determined by measuring across the rosette at the point where the distance was largest. For chlorophyll measurements, 120 seedlings were collected for each genotype or transgenic line. These were pooled and weighed to estimate the freshweight. Chlorophyll was extracted with 80% acetone in 2.5 mM HEPES-KOH (pH 7.5) as described previously (Hackett et al., 2017 ) and chlorophyll amounts were calculated from absorbances measured on a spectrophotometer, as described in Wellburn ( 1994 ). Pairwise Student’s t tests (two-tailed) were used to compare means of rosette diameter and ANOVA to compare the chlorophyll concentration. Chloroplast isolation Chloroplasts from leaf tissue of Arabidopsis thaliana were isolated according to (Bodensohn et al., 2020). Pulse amplitude modulation (PAM) measurements Seedlings cultivated as described above were first dark adapted for 15 min before being placed in the FluorCam 800-C (Photon systems instruments) for measurements. Initially F 0 was recorded for 5 sec with a measuring beam. Thereafter F M was initiated with a saturating flash at 4000 µE. After a relaxation phase of 17 sec actinic light at 150 µE was utilized to assess the photosynthetic adaptation (non-photochemical quenching NPQ) for 5 pulses every 12 sec. This was followed by another measurement in the dark for 3 pulses. Infrared gas analyzer measurements (IRGA) The gas exchange rate of 4week-old plants was measured using a LI-6400XT portable photosynthesis system (Li-Cor Biosciences Inc.). Five individual plants, grown under short-day conditions (8-h light provided by cool white fluorescent bulbs, 100 µM m − 2 s − 1 , 22°C; 16-h dark, 20°C) were used for each experiment. For the light-dependent tests, the whole-plant chamber was set up with the following parameters: 200 µmol/sec flow rate, 20◦C block temperature, 50–70% relative humidity, and 400 µmol/mol [CO2]. For differential light conditions, the plants were dark-exposed (0 PAR) for 30 min and then light-exposed (400 PAR) for 30 min followed by another dark (0 PAR) phase for 30 min. The data was logged every 30 sec. The LI-6400XT portable photosynthesis system (Li-Cor Biosciences Inc.) generates the raw data for the rate of transpiration over time with a default value for the area of the plants. Following the gas exchange rate measurement, ImageJ was used to determine the plant's area (Schindelin et al., 2012 ). The rate of transpiration measurement was automatically standardized on the plant area by adding this value to the raw data from the LI-6400XT device. Furthermore, the time scale was modified in accordance with the experiment. For instance, the graph's x-axis can be modified to show 30 minutes on the negative scale and 60 minutes on the positive scale once the time point 0 is set at 400 PAR light. Data presented are means of at least three leaves from individual plants for each experiment. Experiments were repeated at least three times. Plasmids and plasmid construction for protein expression For the overexpression of GST-tagged mature full-length GET3b (GST-Get3b), the corresponding sequence encoding amino acids (aa) 68–411 was cloned into pGex-4T-3 using the BamHI/SalI restriction sites. The length of the chloroplast transit sequence was predicted by the TargetP-2.0 server ( https://services.healthtech.dtu.dk/services/TargetP-2.0/ ). Constructs for the expression of His-tagged C-terminal regions of ALB3 corresponding to aa 350–462 (Alb3C-His) and ALB4 corresponding to aa 334–499 (Alb4C-His) were described previously (Ackermann et al., 2021 ). Expression and purification of recombinant proteins His-tagged proteins were expressed in E. coli strain Rosetta2(DE3). Cells were cultivated in LB medium at 37 o C to an optical density between 0.6 and 0.8. After induction with 1 mM of isopropyl-β-D-thiogalactopyranosid (IPTG) the cells were grown for additional 3 hours at 30 o C. Collected cells were resuspended in washing buffer (20 mM HEPES, 300 mM NaCl, 40 mM imidazole, 2 mM DTT, pH 8.0). Cells were disrupted by sonification and after centrifugation, the supernatant was either loaded onto a nickel-nitrilotriacetic acid column (GE Healthcare) or incubated with nickel-nitrilotriacetic acid resin (Qiagen). After washing with washing buffer the fusion constructs were eluted with elution buffer (20 mM HEPES, 300 mM NaCl, 250 mM imidazole, 2 mM DTT, pH 8.0). Subsequently, the imidazole was removed from the buffer using PD-10 columns (GE Healthcare). The protein was kept on ice and used directly or was stored at -20°C until usage. GST-Get3b was expressed as described above with the following modifications. Collected bacterial cells were resuspended in 1x PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.3) before sonication. Proteins were purified using glutathione-sepharose (GE Healthcare) and eluted with 10 mM reduced glutathione in 1xPBS buffer. The buffer was exchanged to 1xPBS using PD-10 columns (GE Healthcare). Pull-down assay In vitro pull-down analyses were performed using 50 µg of GST-Get3b and either 20 µg of Alb3C-His or 20 µg of Alb4C-His in a total volume of 120 µl of 1x PBS. Negative control reactions were conducted using GST instead of GST-Get3b or the His-tagged constructs alone. The samples were incubated for 10 min at room temperature. Subsequently, 100 µl of a 50% slurry of glutathione resin (equilibrated in 1x PBS) were added and incubated for 30 min at room temperature. The mixture was transferred to Wizard® Mini Columns (Promega) and centrifuged briefly. The column was washed with 5 ml of 1xPBS buffer using a syringe. After a brief centrifugation of the column the proteins were eluted with 30 µl of 10 mM reduced glutathione in 1xPBS buffer. The load as well as the eluted samples were analyzed by SDS-PAGE and proteins detected by Coomassie staining or Western blotting. Construction of split-ubiquitin plasmids and the split-ubiquitin yeast two-hybrid assays For the split-ubiquitin yeast two-hybrid assay the coding sequence for mature full-length GET3b (aa 68–411) was cloned into pADSL-Nx using the BamHI/SalI restriction sites. All other constructs have been described previously (Bals et al., 2010 ; Pasch et al., 2005 ). The split-ubiquitin assay was done according to (Pasch et al, 2005 ). MS/MS analysis For in-solution mass spectrometry, proteins were digested (León et al., 2013 ; ISD:SDC; AP&PT) and peptides were purified by C18 STAGE-TIPS (Rappsilber et al., 2007 ). Peptides were analyzed using an ultra-HPLC Proxeon EASY-nLC 1000 system coupled online to Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Reversed-phase separation was performed using a 30 cm analytical column (100 µm diameter; DNU-MS (Novak) packed in-house with Reprosil-Pur 120 C18-AQ, 2.4 µm). Mobile-phase solvent A consisted of 0.1% formic acid and 4% acetonitrile in water, and mobile-phase solvent B consisted of 0.1% formic acid in 80% acetonitrile. The flow rate of the gradient was set to 200 nl/min. A 70-min gradient was used (0–40% solvent B within 40 min, 40–100% solvent B within 10 min, 100% solvent B for 10 min, 100–0% solvent B within 5 min and 0% solvent B for 5 min). Data acquisition was performed with the ddMS2 method with the following configuration: For the MS scans, the scan range was set to 250–2,000 m/z at a resolution of 70,000, and the automatic gain control (AGC) target was set to 1 × 10 6 . For the MS/MS scans, Top 13 ions were chosen, the resolution was set to 35,000, the AGC target was set to 1 × 10 5 , the precursor isolation width was 2 Da and the maximum injection time was set to 80 ms. The HCD normalized collision energy was 27%. MaxQuant was used to analyze the LC-MS/MS data (ver 1.5.5.; Cox and Mann, 2008 ) which allowed qualitative and quantitative analysis. The UniProt reference Arabidopsis database (UP000006548) was used for the identification of proteins. Default settings for fixed modifications were used, Dynamic modifications were set: Oxidation for M and Deamidation for NQ. Contaminants were included for peptide detection of a minimum length of 6 amino acids. FDR threshold was set to 0.01. Perseus was used to transform the quantitative data and perform a statistical analysis (Tyanova and Cox, 2018 ). The default settings for a Student’s t-test were utilized for the analysis. For this, independent triplicates corresponding to roughly 80 µg of plastid protein isolated from 8 pots of Arabidopsis thaliana lawn from wild-type and get3b plants were compared. Accession numbers The following Arabidopsis genes were investigated in this study: AT3G10350 (GET3B), AT5G03940 (cpSRP54), AT1G24490 (ALB4/STIC1), and AT2G24020 (STIC2). Results Proteomic characterization of the get3b mutant For the GET3B locus, three T-DNA insertion lines are publicly available (Xing et al., 2017 ). We decided to analyze the get3b-2 mutant (from here on get3b ) with an insertion in the fourth intron of the allele. The line was homozygous for the mutation and had no transcript nor protein detectable by RT-PCR or western blot, respectively (Fig. 1 A-D). To analyze the overall effect of the get3b mutation on chloroplast function, we examined get3b chloroplasts on a proteomic level. By using wild-type and get3b plastids, a label-free technique could be employed in a quantitative manner as previously described (Vermeulen et al., 2008 ). For this, we isolated plastids of the respective lines from 21-day-old A. thaliana seedlings and subjected these to LC-MS/MS. The examination revealed a total of 999 proteinaceous hits. After subtraction of contaminants, 203 were classified as significantly depleted and 105 as enriched in get3b (Fig. 1 E). Intriguingly, not only nuclear encoded proteins, but also plastome encoded proteins were significantly affected. These covered a wide range of biological processes and were manually curated and categorized according to their cellular functions (Fig. 1 F, G). The majority of the significantly depleted hits (33.9%) belonged to the category other metabolic pathways , which included components of the AA synthesis machinery as well as the post-translational modification machinery. Apart from that, chaperone/assembly factors (10.3%), the translational machinery (9.8% ribosomes plus 5.9% transcription/translation ), the photosynthetic machinery (9.8% light reaction plus 2.9% dark reaction ) were mostly affected. Constituents of starch synthesis (5.4%), the plastid redox regulation and translocation (3.4% each), tRNA (2.9%), proteases (2.4%), Fe-S cluster synthesis (1.4%), fatty acids and membranes (0.9% each) were also depleted to a significant extent in get3b (Fig. 1 F). Interestingly, one entire paralogous set of nuclear encoded proteins of the oxygen evolving complex (OEC; Allahverdiyeva et al., 2013 ) was reduced to a significant level. Furthermore, several components of the electron transport chain were also decreased. Additionally, soluble proteins involved in the Benson-Calvin cycle like several subunits of ribulose-1,5-bisphosphat-carboxylase/-oxygenase (RuBisCo) and their assembly factors were also negatively affected as well as STIC2, a factor involved in stromal targeting networks (Bédard et al., 2017 ). Strikingly, GET3B was detectable even though it was significantly depleted (Supplemental table S1 ), suggesting that the T-DNA insertion might lead to a knock-down rather than a knock-out of the respective gene. The majority of the significantly enriched hits were attributed to the light reaction of photosynthesis (39%), followed by other metabolic pathways (21.9%) and components of translocation machineries (12.4%). Additionally, starch synthesis (8.5%), proteases (3.8%), constituents involved in transcription/translation (2.8%), chaperone/assembly factors, redox regulation, Fe-S cluster biogenesis and fatty acid synthesis (1.9% each) as well as elements involved in membrane modulation (0.9%) were elevated to a significant extent (Fig. 1 G). Strikingly, nearly all LHCPs of PSII and a few of PSI were enriched in get3b , while the respective cpSRP targeting machinery was unaffected (Supplemental table S1 ). The comparative proteomic data displayed that the loss or reduction of GET3B has a pervasive effect on the proteome of plastids. Interestingly, the photosynthetic apparatus seemed to be affected the most. get3b mutation affects de novo assembly of photosystem II The data from the comparative proteomics indicated that the light reaction as well as assembly factors of the photosynthetic machinery, like YCF3 and CRR6, were affected in get3b (Fig. 1 E-G, supplemental table S1 ). This prompted us to examine a possible involvement of GET3B function in photosystem assembly. In order to address this, we employed PAM measurements during de-etiolation of A. thaliana seedlings. These measurements exploit the fluorescence solely emitted by PS II, since PS I is a fluorescence quencher (Brettel, 1997 ). We utilized Fv/Fm values as a read out for PS II functionality. By employing this during de-etiolation we were able to assess the extent of de novo formation of PS II. For this, seedlings were germinated and cultivated in vitro on Murashige Skoog (MS) medium (Murashige and Skoog, 1962 ) for 4 days in the dark and in the morning of the fifth day illuminated normally. The course of de-etiolation and concomitant de novo PS II assembly was encompassed by PAM measurements. This occurred 1, 7 and 24 hours after the initial illumination. When cultivated without a carbon source, get3b was significantly hampered in the assembly of PSII in comparison to wild-type after 7 and 24 hours (Fig. 2 A; top panel). In the presence of a carbon source, this difference was only apparent after 24 hours and not to such a high degree of significance, indicating that PS II functionality was still hindered in the absence or reduction of GET3B when grown heterotrophically (Fig. 2 A; bottom panel). As a control we grew the same plates for five days without etiolation and analyzed these fluorometrically. When cultivated autotrophically, get3b was still significantly obstructed in PS II assembly (Fig. 2 B; top panel). Conversely, when grown in the presence of a carbon source, no difference was detectable with PAM measurements (Fig. 2 B; bottom panel). Being able to observe significant differences after five days of growth under normal irradiation, prompted us to analyze 4-day-old seedlings under regular illumination in the absence of sucrose. The corresponding PAM measurements mirrored the previous assessment and confirmed an impaired PSII assembly in get3b (Fig. 2 C). Consistently, get3b also exhibited a significant reduction in chlorophyll content (Fig. 2 D). Taken together these data indicated that, in get3b mutants, not only the initial stages (up to 24 hours) of de novo assembly of PS II were affected but also the later stages (up to 5 days). Additionally, the reduced chlorophyll content of get3b illustrated the obstruction of the photosynthetic machinery during early plant development. get3b mutation affects CO 2 assimilation The proteomic analysis also suggested that components of the dark reaction were affected by the get3b mutation. Interestingly, not only three of four paralogous genes of the nuclear encoded small subunits but also the plastom encoded large subunit of RuBisCo were affected by the reduction or absence of GET3B. Additionally, RuBisCo assembly factors like RbcX, CPN60A1, CPN60B1 and CPN10-2 were significantly affected in get3b (Supplemental table S1 ). In total, these constitute six out of nine components necessary to synthesize RuBisCo in vitro (Aigner et al., 2017 ). In order to address this matter, we analyzed the CO 2 assimilation of A. thaliana seedlings by Infra-red gas analyzer (IRGA; Toro et al., 2019 ). The measurements revealed that get3b assimilated significantly less CO 2 (Fig. 3 ). During the measurements we included a complementation line that was previously used (Xing et al., 2017 ). The complementation line was able to rescue the get3b assimilation phenotype and restore the levels above those of the wild-type (Fig. 3 ). This and the previous data support the results of the proteomic analysis, that GET3B is required for an efficient light as well as dark reaction of photosynthesis. GET3B interacts genetically with components of the STIC pathway (ALB4 and STIC2) Previously we had shown the negative -synergistic effect of combining the get3b and srp54 alleles (Anderson et al., 2021 ). A functional interaction between the two factors also became apparent from our proteomic analysis, in which get3b displayed a significant enrichment of cpSRP substrates (Fig. 1 , supplemental table S1 ). This could be due to a form of compensation for the absence or reduction of GET3B function or the reduction of some stromal proteases which we could also observe in the proteomic data (Supplemental table S1 ). Additionally, the significant depletion of STIC2 in get3b prompted us to analyze the connection between the GET3B-, cpSRP- and the STIC- components. Hence, we utilized the existing single mutants srp54 (Amin et al., 1999 ), stic2 (Bédard et al., 2017 ), alb4 (Gerdes et al., 2006 ) and get3b , and double mutants srp54 get3b (Anderson et al., 2021 ), alb4 srp54 , and alb4 stic2 (Bédard et al., 2017 ), and combined mutant alleles to create the double mutants alb4 get3b and stic2 get3b , and triple mutants alb4 stic2 get3b and alb4 srp54 get3b (Fig. 4 ). To assess the phenotypic consequences of these genetic combinations, we measured the rosette diameter as an indicator of overall plant fitness. Unlike the get3b , alb4 , stic2 single mutants and alb4 stic2 double mutant, the srp54 single mutant and alb4 get3b , stic2 get3b double mutants displayed a significantly smaller rosette diameter compared to the wild-type (Fig. 4 A-F, H, I, M). Strikingly, this phenotypical trait was further exacerbated in the srp54 get3b double mutant and alb4 stic2 get3b triple mutant (Fig. 4 G, J, M). These results illustrate an additive or synergistic interaction between GET3B and STIC components. Notably, the get3b allele in a srp54 background showed a more severe phenotype than in an alb4 stic2 background, suggesting a stronger functional interdependence between GET3B and SRP54. In order to better understand this interdependence as well as the effect of the get3b allele on plant fitness, we analyzed the alb4 srp54 get3b triple mutant. While alb4 srp54 plants were smaller than srp54 plants (Fig. 4 K, M), the addition of the get3b allele resulted in a further reduction in the measured rosette diameter (Fig. 4 L, M). This demonstrated that the combination of the alb4 , srp54 and get3b alleles resulted in an additive detrimental effect on plant fitness. The ATPase function of GET3B is responsible for the growth phenotype In order to investigate the restorative effects of GET3B expression in the alb4 srp54 get3b triple mutant, we complemented the triple mutant by transfecting the plants with a construct previously used (Anderson et al., 2021 ). In the T2 generation, 12 independent lines were identified carrying the ProUB10:GET3B-STREP transgene. Data are shown for 3 randomly chosen lines (Fig. 5 B-C, G). The rosette diameters of the three lines were significantly smaller (lines 2,3) as well as larger (line 1) than the alb4 srp54 double mutant (Fig. 5 A-C, G). However, in all of the lines, rosette diameters were more than three times larger than the alb4 srp54 get3b triple mutant, signifying that the transgene was able to rescue the triple mutant phenotype (Fig. 5 G, H). To determine if the molecular complementation was due to the targeting or holdase function of GET3B, we introduced GET3B transgenes with D124N mutations into the alb4 srp54 get3b background (Fig. 5 D-F). These have also been previously used (Anderson et al., 2021 ) to eliminate the ATPase activity of GET3B (Mateja et al., 2009b ). In the T2 generation 7 independent lines were identified that contained the ProUB10:GET3B (D124N)-STREP transgene. Data are shown for 4 randomly chosen lines. One of the lines (line2) displayed a significantly larger rosette diameter than the triple mutant but all of the lines were approximately three times smaller than the alb4 srp54 double mutant (Fig. 5 D-H). Overall, the transgene with a D124N mutation was unable to rescue the alb4 srp54 get3b phenotype, illustrating that the ATP hydrolysis activity is crucial for complementation (Fig. 5 H). To further investigate the functional consequences of the WT GET3B transgene and the ATP hydrolysis variant, we transfected these constructs into alb4 or stic2 backgrounds. In the T2 generation, we observed distinct phenotypic differences between the untransfected and transgenic lines (Fig. 6 ). Transfection of the WT GET3B transgene into stic2 backgrounds did not result in significant phenotypic differences compared to stic2 plants (Fig. 6 C, D, G). In contrast, transfection of the ATP hydrolysis-deficient GET3B variant into the alb4 background led to a significantly smaller plant size in one of two randomly chosen lines (Fig. 6 E, H). This effect was more pronounced when the transgene was transfected into a stic2 background. Both of the two randomly chosen lines showed highly significant reductions in rosette diameter when compared to stic2 plants (Fig. 6 F, H). These findings show that the loss of ATP hydrolysis of GET3B in conjunction with impairments of the STIC pathway result in stunted growth. This clearly illustrates the importance of both functioning pathways for normal plant growth and chloroplast function. GET3B interacts physically with the C-termini of ALB3 and ALB4 Due to the fact that the alb3 mutant does not survive beyond the seedling stage when germinated on soil (Sundberg et al., 1997 ), we did not include it in our preceding genetic analysis. Still we wanted to analyze the interplay of GET3B with ALB3 as well as ALB4 and verify if the latter two could function as the terminal stromal insertases of the pathway. We were interested in both homologues since these were suggested to engage with similar sets of interactors (Trösch et al., 2015 ). To investigate the interaction between GET3B and ALB3 or ALB4, we recombinantly expressed GET3B C-terminally fused to GST (GST-Get3b) as well as C-terminal fragments of ALB3 and ALB4 N-terminally fused to a His-tag (Alb3C-His and Alb4C-His; Ackermann et al., 2021 ) and performed pulldown experiments (Fig. 7 A, B). We mixed GST-Get3b with Alb3-His as well as GST-Get3b with Alb4C-His and incubated them with Glutathione Sepharose. In order to check for unspecific interactions, Alb3C- and Alb4C-His were incubated with GST and Glutathione Sepharose resin (Fig. 7 A). We observed residual amounts of both Alb3C- and Alb4C-His being pulled down by Glutathione Sepharose and the GST tag, indicating a non-specific interaction with the resin. However, when GST-Get3b was included in the pulldown experiment, we observed a clear enrichment of both Alb3C- and Alb4C-His, suggesting a specific interaction between GET3B and the C-terminus of ALB3 and ALB4 (Fig. 7 B). In order to corroborate the in vitro findings we employed a yeast two-hybrid split ubiquitin assay to further investigate the interaction between GET3B and ALB3 as well as ALB4. We transfected yeast cells with constructs expressing full length ALB3 or ALB4 N-terminally fused to the C-terminus of ubiquitin (Alb3-Cub and Alb4-Cub), along with GET3B fused to the N-terminus of ubiquitin (NubG-Get3b) (Fig. 7 C, D). As controls, we transfected cells with Alg5 fused to the N-terminus of ubiquitin, both with and without the mutation allowing spontaneous reassembly of ubiquitin (Alg5-Nubl and Alg5-NubG, respectively). As additional controls, we tested the C-terminus of ubiquitin N-terminally fused to SEC1 and ALG5 (Sec1-Cub and Alg5-Cub) together with NubG-Get3b (Fig. 7 E). Upon growth on the corresponding dropout medium, we observed robust cell growth in combination with NubG-Get3b and Alb3-Cub as well as Alb4-Cub fusion constructs, indicating a positive interaction (Fig. 7 C, D second column). In contrast, cells co-transfected with the control protein, Alg5-NubG as well as Sec1-Cub did not exhibit significant growth under the same conditions (Fig. 7 E second column). As positive controls, cells expressing Alg5-NubG grew on all media, confirming the functionality of the assay. The combination of the pulldown experiments as well as the yeast two-hybrid split ubiquitin experiment indicated a physical interaction between GET3B and the C-terminus of ALB3 and ALB4. These results together with previous ones (Anderson et al., 2021 ) support the notion that GET3B selectively binds clients and interacts with insertases, potentially facilitating the membrane integration of proteins within chloroplasts. Discussion The findings of this study illuminate the critical role of At GET3B in chloroplast function. We provide new mechanistic insights of its function during photosynthesis and chloroplast biogenesis in Arabidopsis thaliana . Impact of GET3B on chloroplast proteome, PS II assembly and carbon fixation The proteomic analysis provided a comprehensive overview of the downstream effects caused by the absence or reduction of GET3B. This was crucial in identifying the specific proteins and pathways most affected by the get3b mutation, shedding light on the multifaceted role of GET3B in chloroplast biogenesis and function. One of the most striking findings from the proteomic analysis was the global effect on the abundance of plastidic proteins in the get3b mutant (Fig. 1 ). These encompass a wide functional range, suggesting that GET3B has a broad impact on chloroplast proteostasis. The entire photosynthetic apparatus of higher plats contains 16 single pass IMPs (Shi and Schröder, 2004 ): PS II contains 12 of which 7 have a TA topology (Wei et al., 2016 ) PS I comprises two of which none have a TA topology (Pan et al., 2018 ) and the ATP synthase encompasses two of which one shares a TA topology (Hahn et al., 2018 ). Taking the postulated function of GET3B into account as the central element of the stromal TA targeting machinery one would expect the corresponding substrate to be reduced in a genetic background of a knock-out or knock-down. Remarkably, the only single pass IMP that was significantly depleted in get3b was PsbR (Supplemental table S1 ). Together with PsbO, PsbP and PsbQ, PsbR forms the OEC and represents its only IMP (Mishra and Ghanotakis, 1993 ). To date structural analyses of PS II of higher plants were unable to resolve the location and topology of PsbR (Van Bezouwen et al., 2017 ). However, previous biochemical analyses corroborated that the majority of the elongated N-terminus is oriented to the thylakoidal lumen (Ljungberg et al., 1984 ; Ljungberg et al., 1986). If this topology is correct, then no bona fide photosynthetic TA protein was significantly depleted by the absence or reduction of GET3B. This implies that either GET3B is not involved in the targeting of TA proteins or another factor can compensate for its absence (see next section: genetic and physical interactions with STIC component and SRP54). The significant reduction of Fv/Fm values during the de novo assembly of PS II highlight the important role of GET3B in early chloroplast biogenesis. The partial restoration of emitted fluorescence and concomitant PS II functionality in the presence of an external carbon source suggests that GET3B may play a crucial role under autotrophic conditions (Fig. 2 A-C). The observed reduction of Fv/Fm values during de-etiolation could either arise from impediments in OEC assembly/function or the depletion of key photosynthetic assembly factors (Supplemental table S1 ). It is difficult to dissect whether this reduction actually results from an incomplete assembly of PS II or solely an impeded OEC of PS II. Further analyses are necessary to clarify this. Components of chlorophyll biogenesis pathways such as CHL synthase, magnesium chelatase and several assembly factors as well as subunits of RuBisCo were significantly depleted in get3b (Supplemental table S1 ). This directly correlates with the reduced chlorophyll content of young get3b seedlings (Fig. 2 D) as well as the reduced carbon assimilation (Fig. 3 ), respectively. These observations further support the conclusion that GET3B is integral to the proper formation and function of the entire photosynthetic apparatus. However, at this stage it is hard to dissect if these are due to the direct function of GET3B or secondary pleiotropic effects. The proteomic analysis provided critical insight into the molecular underpinnings of the get3b mutation. GET3B appears to be vital for the maintenance of a wide range of chloroplast proteins, particularly those involved in photosynthesis. The broad impact of GET3B on chloroplast proteome underscores its central role in chloroplast biogenesis and function, with potential implications for understanding how chloroplast proteostasis is maintained under various physiological conditions. Genetic and physical interactions with STIC components and SRP54 The negative synergism derived from combining the get3b allele with srp54 , alb4 and stic2 implies an orchestrated network between GET3B and these pathway components probably by ensuring the proper integration and assembly of chloroplast proteins. In particular, the srp54 get3b double mutant displayed a more severe phenotype than either single mutant by itself (Fig. 4 ), signifying that the srp54 mutation acts as a potentiator of the get3b effect or vice versa. This suggests that SRP54 and GET3B either may work together in a single pathway or may work separately in parallel pathways that converge in similar chloroplastidic processes. Interestingly, SRP54 binds solely to the third TMD of LHCPs (High et al., 1997 ). This TMD is the most hydrophobic of all three and fusing it alone to a soluble protein will translocate the fusion protein into the thylakoid membrane (Kohorn and Tobin, 1989 ). Since both SRP54 and GET3B are capable of binding single TMDs, it might be the case that these two proteins are able to partially compensate for the absence of the other, thereby maintaining stromal IMP trafficking when other targeting networks are not fully functional. The incapacity of the ATPase-deficient variant to rescue the growth phenotype in the alb4 srp54 get3b triple mutant (Fig. 5 ) underscores that it is the targeting function of GET3B rather than the potential holdase function which enables its participation in chloroplastidic proteostasis. The observed growth defects of plants expressing the ATPase-defiant variant in alb4 and stic2 backgrounds (Fig. 6 ) further confirm that operative ATPase activity is essential for a fully functioning GET3B in planta. The physical interaction between GET3B and the C-terminal regions of ALB3 and ALB4, as demonstrated by pulldown experiments and yeast two-hybrid assays, provides a mechanistic insight into how GET3B may facilitate the insertion of proteins into the thylakoid membrane. Interestingly, irrespective of in vitro or in vivo , both the stromal exposed C-terminal regions or full length ALB3 and ALB4 interacted with GET3B to a fairly similar degree (Fig. 7 ), indicating less selectivity for the insertases than the cpSRP pathway. The C-terminal regions of ALB3 and ALB4 share two conserved motifs: motif I and motif III, which are not utilized by ALB3 for the interaction with SRP43 (Falk et al., 2010 ). In turn, SRP43 interacts with motifs II and IV of ALB3 which helps it discriminate between the two insertases (Falk et al., 2010 ; Dünschede et al., 2011 ). Recently, a direct physical interaction between STIC2 and motif III of ALB3 and ALB4 was demonstrated (Stolle et al., 2024 ). The plastidic ribosome on the other hand cooperatively binds motif III and IV of ALB3 (Ackermann et al., 2021 ). In future studies, it would be interesting to characterize the GET3B binding site within the C-terminal regions of the Alb proteins and to investigate whether GET3B and STIC2 may engage in competitive or cooperative binding. In summary, the results of this study highlight the role of GET3B in chloroplast biogenesis and function. GET3B appears to be involved in the proper assembly and function of photosynthetic complexes but also for proteostasis in a broader sense within the chloroplast. Its genetic interactions with STIC components and SRP54, as well as its dependence on ATPase activity, suggests a critical role in the integration of membrane proteins. Further studies are necessary to explore the precise mechanistic details of GET3B ’ s interactions with the other targeting components and how they contribute to the overall regulation of chloroplast biogenesis and function. Declarations Acknowledgements The authors would like thank Sarah Friedrich for plant photographs and figure preparation as well as Fabian Fuchs and Silke Funke for experimental support. Conflict of Interest The authors have no conflict of interest to declare. Ethical Compliance The manuscript does not include data or description of human or animal patients . Funding This work was supported by the Deutsche Forschungsgemeinschaft (DFG SCHL 585-10) and the University of Wisconsin-Madison Office of the Vice Chancellor for Research and Graduate Education. Competing interests The authors have no relevant financial or non-financial interests to disclose. Author contribution Conceptualization: [Uwe Bodensohn, Danja Schuenemann, Donna Fernandez and Enrico Schleiff]; Genetic analyses: [Donna Fernandez]; Interaction analyses: [Beatrix Dünschede, Chiara Kuhlmann]; IRGA measurements: [Khushbu Kumari]; LC-MS/MS [Roman Ladig]; Physiological experiments: [Uwe Bodensohn]; Writing – original draft preparation: [Uwe Bodensohn]; Writing – review and editing: [Uwe Bodensohn, Danja Schuenemann, Donna Fernandez]; Funding acquisition: [Enrico Schleiff, Donna Fernandez]; Supervision: [Uwe Bodensohn, Danja Schuenemann, Donna Fernandez, Christopher Grefen, Enrico Schleiff] Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Ackermann, B., Dünschede, B., Pietzenuk, B., Justesen, B.H., Krämer, U., Hofmann, E., Günther Pomorski, T., and Schünemann, D. (2021). Chloroplast Ribosomes Interact With the Insertase Alb3 in the Thylakoid Membrane. 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Supplementary Files SupplementalFileBodensohnetal..docx Cite Share Download PDF Status: Published Journal Publication published 29 Apr, 2025 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Accept 10 Apr, 2025 Reviewers agreed at journal 20 Mar, 2025 Reviewers invited by journal 20 Mar, 2025 Editor assigned by journal 20 Mar, 2025 First submitted to journal 17 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6212458","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431783732,"identity":"e0cd95eb-4d47-4cd6-91cf-f8db0560ddbc","order_by":0,"name":"Uwe Sakamuzi Bodensohn","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4698-3757","institution":"Goethe-Universitat Frankfurt am Main","correspondingAuthor":true,"prefix":"","firstName":"Uwe","middleName":"Sakamuzi","lastName":"Bodensohn","suffix":""},{"id":431783733,"identity":"fe78ae8e-e440-4a0a-b518-e4426da0accc","order_by":1,"name":"Beatrix Dünschede","email":"","orcid":"","institution":"Ruhr-Universität Bochum: Ruhr-Universitat Bochum","correspondingAuthor":false,"prefix":"","firstName":"Beatrix","middleName":"","lastName":"Dünschede","suffix":""},{"id":431783734,"identity":"14b5422c-a157-44c3-aabd-224632b30df0","order_by":2,"name":"Chiara Kuhlmann","email":"","orcid":"","institution":"Ruhr University Bochum: Ruhr-Universitat Bochum","correspondingAuthor":false,"prefix":"","firstName":"Chiara","middleName":"","lastName":"Kuhlmann","suffix":""},{"id":431783735,"identity":"35ebb6d4-fb18-4490-bff2-2c11e52ce685","order_by":3,"name":"Khushbu Kumari","email":"","orcid":"","institution":"Ruhr-Universitat Bochum","correspondingAuthor":false,"prefix":"","firstName":"Khushbu","middleName":"","lastName":"Kumari","suffix":""},{"id":431783736,"identity":"4eeaf64f-6c7e-464b-a6a8-7ea57ad01971","order_by":4,"name":"Roman Ladig","email":"","orcid":"","institution":"German Cancer Research Centre: Deutsches Krebsforschungszentrum","correspondingAuthor":false,"prefix":"","firstName":"Roman","middleName":"","lastName":"Ladig","suffix":""},{"id":431783737,"identity":"a0c93dcc-3b1d-4331-9d2a-12659f5a3083","order_by":5,"name":"Chrisopher Grefen","email":"","orcid":"","institution":"Ruhr-Universität Bochum: Ruhr-Universitat Bochum","correspondingAuthor":false,"prefix":"","firstName":"Chrisopher","middleName":"","lastName":"Grefen","suffix":""},{"id":431783738,"identity":"dac4160b-51a6-4694-98ed-a197ea2fca2c","order_by":6,"name":"Enrico Schleiff","email":"","orcid":"","institution":"Goethe University Frankfurt: Goethe-Universitat Frankfurt am Main","correspondingAuthor":false,"prefix":"","firstName":"Enrico","middleName":"","lastName":"Schleiff","suffix":""},{"id":431783739,"identity":"39b6d44f-e4a3-4352-b850-680490cd3b0a","order_by":7,"name":"Donna Fernandez","email":"","orcid":"","institution":"University of Wisconsin-Madison","correspondingAuthor":false,"prefix":"","firstName":"Donna","middleName":"","lastName":"Fernandez","suffix":""},{"id":431783740,"identity":"c6aa2460-b2d3-4cf3-856a-afe16745c322","order_by":8,"name":"Danja Schünemann","email":"","orcid":"","institution":"Ruhr-Universität Bochum: Ruhr-Universitat Bochum","correspondingAuthor":false,"prefix":"","firstName":"Danja","middleName":"","lastName":"Schünemann","suffix":""}],"badges":[],"createdAt":"2025-03-12 13:24:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6212458/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6212458/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-025-03500-2","type":"published","date":"2025-04-29T15:57:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79595434,"identity":"b1bca555-310e-45d0-b54a-6d269b9f76ec","added_by":"auto","created_at":"2025-03-31 14:05:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":340472,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eget3b \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutation and the downstream effects on the chloroplast proteome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eGene model of \u003cem\u003eGET3B\u003c/em\u003eand the corresponding T-DNA insertion line analyzed in this study.\u003cstrong\u003e (B)\u003c/strong\u003e Genotyping via PCR of \u003cem\u003eget3b\u003c/em\u003e. The locations of the used primers are indicated in A as arrowheads. \u003cstrong\u003e(C)\u003c/strong\u003eSemi RT-PCR to validate the missing transcript. \u003cstrong\u003e(D)\u003c/strong\u003e Western blot analysis of total protein lysates. Wild-type and \u003cem\u003eget3b\u003c/em\u003e plastids were isolated and subjected to LC-MS/MS followed by label free quantification. \u003cstrong\u003e(E)\u003c/strong\u003e Statistical analysis of proteinaceous hits that were significantly effected in \u003cem\u003eget3b\u003c/em\u003e chloroplasts (see Supplemental table S1 for more details). The significantly depleted \u003cstrong\u003e(F)\u003c/strong\u003e and significantly enriched \u003cstrong\u003e(G)\u003c/strong\u003eproteinaceous hits were categorized according to their cellular function. The color coding in E corresponds to those in F and G.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6212458/v1/fbff238a0cb86fcc7f6a8387.png"},{"id":79595445,"identity":"265ce120-3bd5-4f1f-a555-f49cceac7b62","added_by":"auto","created_at":"2025-03-31 14:05:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":219597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGET3B function is involved in the assembly of photosystem (PS) II\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Seedlings were germinated and cultivated under etiolating conditions for 5 days on 0% sucrose (top) or 1 % sucrose (bottom) for 5 days before being normally illuminated and subjected to pulse amplitude modulation (PAM) measurements. The Fv/Fm ratio (maximum quantum yield) was determined after 1, 7, 24 hours to monitor PS II assembly. \u003cstrong\u003e(B)\u003c/strong\u003e Seedlings were grown under normal illumination on 0 % sucrose (top) and 1 % sucrose (bottom). After 5 days these were subjected to PAM measurements. \u003cstrong\u003e(C)\u003c/strong\u003e Seedlings were cultivated under normal conditions for 4 days and either subjected to PAM measurements or \u003cstrong\u003e(D) \u003c/strong\u003eused for chlorophyll isolation. Statistical analyses for (A) and (B) were performed using the Student’s t-test, for three independent experiments with 40 seedlings per experiment. Statistical analyses for (C) were performed with ANOVA for 15 independent experiments with 40 seedlings per experiment as well as (D) for 3 experiments with 120 seedlings per experiment.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6212458/v1/4b40d8289461ea6746f00517.png"},{"id":79595438,"identity":"1780094f-b531-44ff-91f5-34f3fa504767","added_by":"auto","created_at":"2025-03-31 14:05:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":171418,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGET3B function is involved in CO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e assimilation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4-week-old plants of the depicted lines were subjected to infrared gas analyzer (IRGA) measurements. Data points represent the mean of 4 independent measurements. Asterisks denote significant differences (one way anova ***P\u0026lt;0.001). N.S. : not significant\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6212458/v1/4bf2d32f0f37f0389a447026.png"},{"id":79595447,"identity":"fdaaa9f1-d685-4eea-911f-abc6282f5daf","added_by":"auto","created_at":"2025-03-31 14:05:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":837135,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic interactions are seen when mutations in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGET3B\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eare combined with mutations affecting \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eALB4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and/or \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSTIC2\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePlants were grown in 12-h light/12-h dark conditions for 4 weeks. Top row: Images of \u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003estic2\u003c/em\u003e, \u003cstrong\u003e(B) \u003c/strong\u003e\u003cem\u003ealb4\u003c/em\u003e \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003esrp54,\u003c/em\u003e \u003cstrong\u003e(D)\u003c/strong\u003e Columbia wild-type plants. Middle row: Phenotypes when \u003cem\u003eget3b\u003c/em\u003e mutations were introduced: \u003cstrong\u003e(E)\u003c/strong\u003e \u003cem\u003estic2 get3b\u003c/em\u003e, \u003cstrong\u003e(F)\u003c/strong\u003e \u003cem\u003ealb4\u003c/em\u003e \u003cem\u003eget3b\u003c/em\u003e, \u003cstrong\u003e(G)\u003c/strong\u003e \u003cem\u003esrp54 get3b\u003c/em\u003e, \u003cstrong\u003e(H)\u003c/strong\u003e \u003cem\u003eget3b\u003c/em\u003e plants. Bottom row: \u003cstrong\u003e(I)\u003c/strong\u003e\u003cem\u003ealb4 stic2, \u003c/em\u003e\u003cstrong\u003e(J) \u003c/strong\u003e\u003cem\u003ealb4 stic2 get3b\u003c/em\u003e, \u003cstrong\u003e(K)\u003c/strong\u003e \u003cem\u003ealb4 srp54\u003c/em\u003e, \u003cstrong\u003e(L) \u003c/strong\u003e\u003cem\u003ealb4 srp54 get3b\u003c/em\u003e plants.\u003cem\u003e \u003c/em\u003eBlack areas were added so that plants would be shown at equal magnification. Bars= 1 cm. \u003cstrong\u003e(M)\u003c/strong\u003e Rosette diameters of plants with different genotypes in the absence (left) or presence (right) of \u003cem\u003esrp54\u003c/em\u003emutations. Data are presented as means \u003cu\u003e+\u003c/u\u003e SE (n= 2-10 individual plants). Asterisks designate significant differences (Student’s \u003cem\u003et\u003c/em\u003e test, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) from wild-type or between the samples indicated with brackets.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6212458/v1/366c0bbf971ed802af012019.png"},{"id":79595440,"identity":"46f7c23d-559b-47b6-92a3-ddd0e8d544c0","added_by":"auto","created_at":"2025-03-31 14:05:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":371487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransgenes encoding wild-type GET3B can complement alb4 srp54 get3b triple mutants, but not transgenes encoding GET3B with a D124N mutation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlants were grown in 12-h light/12-h dark conditions for 4 weeks. Top row: Images of \u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003ealb4\u003c/em\u003e \u003cem\u003esrp54\u003c/em\u003e plants, and \u003cstrong\u003e(B, C)\u003c/strong\u003e two independent lines of transgenic plants carrying the ProUBI10:GET3B-STREP transgene in an \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e background. Bottom row: Images of \u003cstrong\u003e(D)\u003c/strong\u003e \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e plants, and \u003cstrong\u003e(E, F)\u003c/strong\u003e two independent lines of transgenic plants carrying the ProUBI10:GET3B(D124N)-STREP transgene in an \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e background. Bars= 1 cm. \u003cstrong\u003e(G)\u003c/strong\u003eRosette diameters of \u003cem\u003ealb4 srp54\u003c/em\u003eplants and three independent lines of \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e plants carrying the ProUBI10:GET3B-STREP transgene. \u003cstrong\u003e(H)\u003c/strong\u003e Rosette diameters of untransformed (T0) \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e plants and four independent lines of \u003cem\u003ealb4 srp54 get3b\u003c/em\u003eplants carrying the ProUBI10:GET3B(D124N)-STREP transgene. Data are presented as means + SE (n= 8-10 individual plants in G, n= 3-8 individual plants in H). Asterisks designate significant differences (Student’s t test, *P\u0026lt;0.05, **P\u0026lt;0.01) between the transgenic plants and either \u003cem\u003ealb4 srp54\u003c/em\u003e plants (G) or \u003cem\u003ealb4 srp54\u003c/em\u003e get3b plants (H).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6212458/v1/a9c425f937ef4fada8ddaf68.png"},{"id":79595446,"identity":"736a4563-d80f-47d2-a74d-739cd3122ebd","added_by":"auto","created_at":"2025-03-31 14:05:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":952073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of transgenes encoding GET3B with a D124N mutation leads to reductions in plant size in alb4 or stic2 genetic backgrounds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlants were grown in 12-h light/12-h dark conditions for 4 weeks. Top row: Images of \u003cstrong\u003e(A)\u003c/strong\u003e Columbia wild-type, \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003ealb4\u003c/em\u003e, and \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003estic2\u003c/em\u003e plants. Bottom row: Transgenic plants carrying the \u003cstrong\u003e(D)\u003c/strong\u003e ProUBI10:GET3B-STREP transgene in a \u003cem\u003estic2\u003c/em\u003e background, \u003cstrong\u003e(E)\u003c/strong\u003e ProUBI10:GET3B(D124N)-STREP transgene in an \u003cem\u003ealb4\u003c/em\u003e background and \u003cstrong\u003e(F)\u003c/strong\u003e ProUBI10:GET3B(D124N)-STREP transgene in a \u003cem\u003estic2\u003c/em\u003e background. Bars= 1 cm. \u003cstrong\u003e(G)\u003c/strong\u003e Rosette diameters of untransformed (T0) \u003cem\u003estic2\u003c/em\u003e plants and two independent lines of transgenic \u003cem\u003estic2\u003c/em\u003eplants carrying the ProUBI10:GET3B-STREP transgene. \u003cstrong\u003e(H)\u003c/strong\u003e Rosette diameters of untransformed (T0) alb4 (left) or \u003cem\u003estic2\u003c/em\u003e (right) plants and two independent lines each of transgenic \u003cem\u003ealb4\u003c/em\u003e or \u003cem\u003estic2\u003c/em\u003e plants carrying the ProUBI10:GET3B(D124N)-STREP transgene. Data are presented as means + SE (n= 10-14 individual plants in G, n= 10-15 individual plants in H). Asterisks indicate significant differences (Student’s t test, *P\u0026lt;0.05, **P\u0026lt;0.01) between the transgenic plants and untransformed (T0) plants of the same genotype.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6212458/v1/bf217c71301b7c6fc3a6b3c8.png"},{"id":79596718,"identity":"6ef0f8f6-b256-4b46-9e25-753990f41123","added_by":"auto","created_at":"2025-03-31 14:21:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":429270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGet3b interacts with the thylakoid membrane proteins Alb3 and Alb4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e In vitro pull-down assays were performed with recombinant GST-tagged mature Get3b (GST-Get3b) and the His-tagged C-terminus of Alb3 (Alb3C-His) or Alb4 (Alb4C-His). Samples containing GST and the C-termini, or the C-termini alone served as negative controls. Approximately 4% of the load samples were applied to SDS-PAGE analysis and stained with Coomassie. \u003cstrong\u003e(B)\u003c/strong\u003e 10% of the eluates of the pull-down assay were subjected to immunoblot analysis using antibodies specific for Alb3, Alb4, or GST. The asterisks indicate truncated forms of the GST-Get3b. The closed circle indicates a nonspecific reaction of the anti-Alb4 antibody with GST. \u003cstrong\u003e(C)\u003c/strong\u003eSplit-ubiquitin yeast two-hybrid assay using mature Get3b and mature full-length Alb3. Alb3 was used as fusion with the C-terminal half of ubiquitin (Cub) at its C-terminus (Alb3-Cub). Get3b was used as fusion with a modified N-terminal half of ubiquitin (NubG) at its N-terminus (NubG-Get3b). The unrelated endoplasmic reticulum membrane protein Alg5 fused to wild-type Nub (Alg5-NubI) and NubG (Alg5-NubG) served as positive and negative controls for the Cub-fusion construct, respectively. DSY-1 yeast colonies were first plated on permissive medium (−LT, lacking Leu and Trp) and on selective medium (−LTH, lacking Leu, Trp, and His). \u003cstrong\u003e(D)\u003c/strong\u003e Same assay as in (C) but using mature full-length Alb4 as bait. \u003cstrong\u003e(E)\u003c/strong\u003e Split-ubiquitin interaction tests of SecY1-Cub with NubG-Get3b and of Alg5-Cub with NubG-Get3b as negative control.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6212458/v1/74ebab8075dc2ff3e260a408.png"},{"id":81987914,"identity":"7089ac72-33d4-4a12-9596-32bb1cbdd2aa","added_by":"auto","created_at":"2025-05-05 16:06:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6323370,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6212458/v1/011195b4-0b71-4003-a9f9-cc15e1f8dc27.pdf"},{"id":79595443,"identity":"04580825-4349-4327-90e2-b1f39780b7ae","added_by":"auto","created_at":"2025-03-31 14:05:36","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":40787,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFileBodensohnetal..docx","url":"https://assets-eu.researchsquare.com/files/rs-6212458/v1/118f2422bbaa3669327ecbfa.docx"}],"financialInterests":"","formattedTitle":"GET3B is involved in chloroplast biogenesis and interacts with the thylakoidal ALB3 and ALB4 insertases","fulltext":[{"header":"Key message ","content":"\u003cp\u003eProteomic, functional physiological analyses of \u003cem\u003eget3b\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003emutant plants highlight GET3B\u0026rsquo;s role in chloroplast function. Genetic and interaction analyses indicate \u003cem\u003eget3b\u003c/em\u003e and \u003cem\u003esrp54\u003c/em\u003e as mutual potentiators that might share terminal insertases.\u0026nbsp;\u003c/p\u003e\n"},{"header":"Introduction","content":"\u003cp\u003eIntrinsic membrane proteins (IMPs) represent an extremely diversified group that represents roughly 30% of the cellular proteome (Wallin and Von Heijne, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Tail anchored (TA) proteins share a simple type II topology with a cytosolically exposed N-terminus and a single C-terminal transmembrane domain (TMD) within the last 50 amino acids (AAs) of the polypeptide stretch. Even though they constitute 3\u0026ndash;5% of eukaryotic IMPs (Kalbfleisch et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), TA proteins fulfill a multitude of essential biological functions like apoptosis (Jiang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), vesicle transport (van Berkel et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and organelle biogenesis (Sommer et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The guided entry of TA proteins (GET) pathway facilitates the post-translational targeting of this subset of IMPs. After translation is terminated, the pretargeting complex captures the TA protein and hands it over to the central ATPase of the GET pathway, Get3 (Wang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Thereafter the TA protein is passed on to its cognate receptor, which functions as an insertase (Wang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For reasons of simplicity, we will generally refer to \u0026ldquo;Get\u0026rdquo; factors and plant specific elements will be denoted in all capital letters, in agreement with \u003cem\u003eArabidopsis thaliana\u003c/em\u003e nomenclature.\u003c/p\u003e \u003cp\u003eA characteristic trait of Get3 is the formation of a symmetric homodimer equipped with a static ATPase domain and a flexible α-helical domain that is sensitive to nucleotide binding. This nucleotide binding state induces the formation of a hydrophobic groove or chamber capable of binding TA substrate (Mateja et al., 2009; Bozkurt et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Mateja et., 2015, Barlow et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In a cellular context, Get3 also has a secondary function in eukaryotes by acting as an ATP-independent holdase under energy limiting conditions and oxidative stress (Powis et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Voth et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Get3 and its orthologues TRC40/ArsA are highly conserved in all domains of life and have diversified into three clades, namely clade a, clade bc and clade d (Xing et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Barlow et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In contrast to metazoans and fungi, which solely contain clade a Get3 homologues, photosynthetic organisms contain multiple Get3 homologues from all clades (Xing et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bodensohn et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Barlow et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The three clades differ in their amino acid (AA) composition, which results in different domain architectures. In the majority of the cases clade a proteins have a cytosolic and clade bc an organellar localization (Xing et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bodensohn et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Depending on the prokaryotic or eukaryotic origin of a photosynthetic organism, clade d proteins have a cytosolic or organellar localization, respectively (Barlow et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eArabidopsis thaliana\u003c/em\u003e has four Get3 homologues, namely \u003cem\u003eAt\u003c/em\u003eGET3A, \u003cem\u003eAt\u003c/em\u003eGET3B, \u003cem\u003eAt\u003c/em\u003eGET3C and \u003cem\u003eAt\u003c/em\u003eGET3D localized to cytosol, chloroplasts, mitochondria and chloroplasts, respectively (Duncan et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Xing et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bodensohn et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Barlow et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Loss of function mutations in any of the clade a or clade bc orthologues renders the resulting plant largely indistinguishable from the wild-type (Xing et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This is also the case in yeast and nematodes in which the deletion of Get3 leads to conditional growth defects (Schuldiner et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tseng et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In mammals however, the disruption of TRC40 is embryo lethal (Mukhopadhyay et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn photosynthetic eukaryotes, plastids constitute one of the most complex systems in terms of protein sorting. 95% of their proteome is encoded by nuclear genes and precursor proteins are synthesized on cytosolic ribosomes to be imported post-translationally. This is facilitated by two multimeric translocons embedded in the outer (TOC) and inner (TIC) envelope that form a continuum from the cytosol, through the intermembrane space (IMS) into the stroma (Liu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Once arrived in the stroma, precursor proteins can be directed to numerous translocation machineries imbedded within the inner envelope or thylakoid membrane. This leads to six different sub organellar localizations that each can individually be accessed with different sets of protein targeting networks (Jarvis and L\u0026oacute;pez-Juez, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Taking the pivotal role of plastids into account, the targeting and translocation networks must operate with high fidelity to ensure proper organellar biogenesis, repair and functionality. This is reflected by the deficiencies in plant fitness that arise when components of these are disrupted (Bauer et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Amin et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Pilgrim et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Sundberg et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne intensively studied stromal targeting pathway is the post-translational cpSRP pathway, which targets a single population of IMPs, namely light harvesting chlorophyll binding proteins, to the thylakoid membrane (Schuenemann et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Ziehe et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). CpSRP is composed of cpSRP54 (henceforth referred to as SRP54), the plastidic homologue of a highly conserved subunit of the cytosolic SRP, and a plant specific targeting factor, cpSRP43 (SRP43 from here on). SRP54 can also interact with the plastidic 70S ribosome and mediates co-translational transport of substrate proteins to its respective insertase, ALB3 and the SecYE translocon (Hristou et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Moore et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe terminal insertase ALB3 belongs to the Oxa1 superfamily of membrane protein biogenesis factors which also includes the ER resident Get1 (Anghel et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In plants, an additional homologue of ALB3 exists, ALB4 (Gerdes et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). ALB4 is involved in the assembly and stabilization of ATP synthase intermediates (Benz et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Furthermore, subsequent analyses displayed that ALB4 works at least partially together with ALB3 and participates in the biogenesis of a subset of thylakoid membrane proteins. (Tr\u0026ouml;sch et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eALB4 also interacts with STIC2, a protein identified in a genetic screen for the suppressors of chlorotic \u003cem\u003etic40\u003c/em\u003e, an \u003cem\u003eA. thaliana\u003c/em\u003e mutant lacking the chloroplast inner envelope protein TIC40. The STIC system is described as a two component network composed of STIC1 (ALB4) and STIC2, which acts together in a shared pathway with cpSRP54 and cpFtsY (B\u0026eacute;dard et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). STIC2 shares homology with the bacterial YbaB,(Link et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Recently, STIC2, was identified as a factor associated with plastidic ribosomes translating the photosystem (PS) II reaction center protein D1, is likely involved in co-translational sorting of other plastid-encoded multi-pass IMPs (Stolle et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a previous study, we provided evidence that the \u003cem\u003eAt\u003c/em\u003eGET3B can selectively bind SECE1 and shows a genetic interaction with components of the cpSRP system. Furthermore, mutant plants showed altered levels of photosynthesis-related proteins (Anderson et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This prompted us to investigate if GET3B interacts with constituents of the cpSRP and STIC systems, with an emphasis on their cognate receptors ALB3 and ALB4, respectively. We hypothesized that GET3B might play a crucial role in the targeting and membrane integration of specific proteins within chloroplasts, thereby contributing to the biogenesis and functionality of chloroplast membranes. To address this hypothesis, we employed a combination of biochemical, molecular, fluorometric and genetic approaches to provide evidence that GET3B physically interacts with the C-terminus of ALB3 as well as ALB4 and genetically interacts with components of the STIC and cpSRP pathways, and that its disruption has an effect on chloroplast biogenesis and function.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and plant growth\u003c/h2\u003e \u003cp\u003e \u003cem\u003eArabidopsis thaliana\u003c/em\u003e seeds were surface sterilized and sown on 0.8% (w/v) agar plates containing \u0026frac12; strength Murashige and Skoog media (Caisson Labs), 50mM MES-KOH, pH 5.7, and 1% (w/v) sucrose. Plants used for fluorometric measurements were germinated and cultivated on MS plates without sucrose. Following 2\u0026ndash;3 days of stratification at 4\u0026deg;C, the plates were exposed to 100 \u0026micro;E m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of light provided by cool white (4100K) fluorescent bulbs on a 12 h light/12 h dark cycle for 7\u0026ndash;14 days at 22\u0026deg;C, at which point seedlings were transplanted to a soil-less mix (Fafard Germinating Mix, Sungro Horticulture), watered with 10% Hoagland\u0026rsquo;s solution (Hoagland and Arnon, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1950\u003c/span\u003e), and grown under the same conditions as the plates.\u003c/p\u003e \u003cp\u003eColumbia-0 ecotype was used as wild-type for all pictures and measurements in this study. Seeds for the T-DNA insertional mutants were acquired from the Arabidopsis Biological Resource Center (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://abrc.osu.edu/\u003c/span\u003e\u003cspan address=\"https://abrc.osu.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Mutant alleles previously characterized as resulting in loss of function were used for the genetic crosses: \u003cem\u003eget3b-2\u003c/em\u003e ( SALK_017702C, Xing et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003ecpsrp54-3\u003c/em\u003e (WiscDsLox289_292B14, Yu et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), \u003cem\u003ealb4-1\u003c/em\u003e (SALK_136199, B\u0026eacute;dard et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003estic2-3\u003c/em\u003e (SALK_001500, B\u0026eacute;dard et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e ) and \u003cem\u003estic2-4\u003c/em\u003e (WiscDsLox445D01, B\u0026eacute;dard et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The two \u003cem\u003estic2\u003c/em\u003e alleles behaved similarly; therefore, we refer to \u003cem\u003estic2\u003c/em\u003e mutants without the allele number in other sections of the paper. Lines carrying the mutant alleles were identified by PCR genotyping using the genotyping primers listed in Supplemental Table S2.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeneration of transgenic plants\u003c/h3\u003e\n\u003cp\u003eGeneration of the constructs used for moderate overexpression and genetic complementation experiments was previously described (Anderson et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The \u003cem\u003eProUBI10:GET3B-STREP\u003c/em\u003e construct contains the promoter for the \u003cem\u003eUBIQUITIN 10\u003c/em\u003e gene (Norris et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), genomic sequence for the coding region of \u003cem\u003eGET3B\u003c/em\u003e, sequence encoding a C-terminal 1X STREP tag, and the terminator of the \u003cem\u003eNOS\u003c/em\u003e (\u003cem\u003enopaline synthase\u003c/em\u003e) gene. For the \u003cem\u003eProUBI10:GET3B(D124N)-STREP\u003c/em\u003e construct, the D124N mutation was introduced into the \u003cem\u003eProUBI10:GET3B-STREP\u003c/em\u003e construct by site-directed mutagenesis. The Arabidopsis plants transformed for the genetic complementation experiments were homozygous for the \u003cem\u003ealb4\u003c/em\u003e and \u003cem\u003eget3b\u003c/em\u003e alleles, and heterozygous for the \u003cem\u003esrp54\u003c/em\u003e allele. Plants transformed for moderate overexpression studies were homozygous for either the \u003cem\u003ealb4\u003c/em\u003e or \u003cem\u003estic2-4\u003c/em\u003e allele. The plant transformation procedure was previously described (Anderson et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and transformants were selected in the T1 generation based on gentamycin resistance. Transformants containing mutant alleles as well as the introduced transgene were identified by PCR genotyping using primers listed in Supplemental Table S2. Genetic complementation was assessed in the T2 generation based on the phenotype of plants that were homozygous for \u003cem\u003ealb4\u003c/em\u003e, \u003cem\u003eget3b\u003c/em\u003e and \u003cem\u003esrp54\u003c/em\u003e and carried the introduced transgene. The effects of moderate overexpression in \u003cem\u003ealb4\u003c/em\u003e or \u003cem\u003estic2\u003c/em\u003e homozygous backgrounds were assessed in either the T2 or T3 generations.\u003c/p\u003e\n\u003ch3\u003eRosette measurements\u003c/h3\u003e\n\u003cp\u003eFor measurements of rosette diameters and chlorophyll content, plants with specific genotypes were grown for approximately 4 weeks under the conditions described above. Rosette diameter was determined by measuring across the rosette at the point where the distance was largest. For chlorophyll measurements, 120 seedlings were collected for each genotype or transgenic line. These were pooled and weighed to estimate the freshweight. Chlorophyll was extracted with 80% acetone in 2.5 mM HEPES-KOH (pH 7.5) as described previously (Hackett et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and chlorophyll amounts were calculated from absorbances measured on a spectrophotometer, as described in Wellburn (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Pairwise Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e tests (two-tailed) were used to compare means of rosette diameter and ANOVA to compare the chlorophyll concentration.\u003c/p\u003e\n\u003ch3\u003eChloroplast isolation\u003c/h3\u003e\n\u003cp\u003eChloroplasts from leaf tissue of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were isolated according to (Bodensohn et al., 2020).\u003c/p\u003e\n\u003ch3\u003ePulse amplitude modulation (PAM) measurements\u003c/h3\u003e\n\u003cp\u003eSeedlings cultivated as described above were first dark adapted for 15 min before being placed in the FluorCam 800-C (Photon systems instruments) for measurements. Initially F\u003csub\u003e0\u003c/sub\u003e was recorded for 5 sec with a measuring beam. Thereafter F\u003csub\u003eM\u003c/sub\u003e was initiated with a saturating flash at 4000 \u0026micro;E. After a relaxation phase of 17 sec actinic light at 150 \u0026micro;E was utilized to assess the photosynthetic adaptation (non-photochemical quenching NPQ) for 5 pulses every 12 sec. This was followed by another measurement in the dark for 3 pulses.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInfrared gas analyzer measurements (IRGA)\u003c/h2\u003e \u003cp\u003eThe gas exchange rate of 4week-old plants was measured using a LI-6400XT portable photosynthesis system (Li-Cor Biosciences Inc.). Five individual plants, grown under short-day conditions (8-h light provided by cool white fluorescent bulbs, 100 \u0026micro;M m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 22\u0026deg;C; 16-h dark, 20\u0026deg;C) were used for each experiment. For the light-dependent tests, the whole-plant chamber was set up with the following parameters: 200 \u0026micro;mol/sec flow rate, 20◦C block temperature, 50\u0026ndash;70% relative humidity, and 400 \u0026micro;mol/mol [CO2]. For differential light conditions, the plants were dark-exposed (0 PAR) for 30 min and then light-exposed (400 PAR) for 30 min followed by another dark (0 PAR) phase for 30 min. The data was logged every 30 sec. The LI-6400XT portable photosynthesis system (Li-Cor Biosciences Inc.) generates the raw data for the rate of transpiration over time with a default value for the area of the plants. Following the gas exchange rate measurement, ImageJ was used to determine the plant's area (Schindelin et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The rate of transpiration measurement was automatically standardized on the plant area by adding this value to the raw data from the LI-6400XT device. Furthermore, the time scale was modified in accordance with the experiment. For instance, the graph's x-axis can be modified to show 30 minutes on the negative scale and 60 minutes on the positive scale once the time point 0 is set at 400 PAR light. Data presented are means of at least three leaves from individual plants for each experiment. Experiments were repeated at least three times.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmids and plasmid construction for protein expression\u003c/h3\u003e\n\u003cp\u003eFor the overexpression of GST-tagged mature full-length GET3b (GST-Get3b), the corresponding sequence encoding amino acids (aa) 68\u0026ndash;411 was cloned into pGex-4T-3 using the BamHI/SalI restriction sites. The length of the chloroplast transit sequence was predicted by the TargetP-2.0 server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/TargetP-2.0/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/TargetP-2.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConstructs for the expression of His-tagged C-terminal regions of ALB3 corresponding to aa 350\u0026ndash;462 (Alb3C-His) and ALB4 corresponding to aa 334\u0026ndash;499 (Alb4C-His) were described previously (Ackermann et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eExpression and purification of recombinant proteins\u003c/h3\u003e\n\u003cp\u003eHis-tagged proteins were expressed in \u003cem\u003eE. coli\u003c/em\u003e strain Rosetta2(DE3). Cells were cultivated in LB medium at 37 \u003csup\u003eo\u003c/sup\u003eC to an optical density between 0.6 and 0.8. After induction with 1 mM of isopropyl-β-D-thiogalactopyranosid (IPTG) the cells were grown for additional 3 hours at 30 \u003csup\u003eo\u003c/sup\u003eC. Collected cells were resuspended in washing buffer (20 mM HEPES, 300 mM NaCl, 40 mM imidazole, 2 mM DTT, pH 8.0). Cells were disrupted by sonification and after centrifugation, the supernatant was either loaded onto a nickel-nitrilotriacetic acid column (GE Healthcare) or incubated with nickel-nitrilotriacetic acid resin (Qiagen). After washing with washing buffer the fusion constructs were eluted with elution buffer (20 mM HEPES, 300 mM NaCl, 250 mM imidazole, 2 mM DTT, pH 8.0). Subsequently, the imidazole was removed from the buffer using PD-10 columns (GE Healthcare). The protein was kept on ice and used directly or was stored at -20\u0026deg;C until usage.\u003c/p\u003e \u003cp\u003eGST-Get3b was expressed as described above with the following modifications. Collected bacterial cells were resuspended in 1x PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.8 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, pH 7.3) before sonication. Proteins were purified using glutathione-sepharose (GE Healthcare) and eluted with 10 mM reduced glutathione in 1xPBS buffer. The buffer was exchanged to 1xPBS using PD-10 columns (GE Healthcare).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePull-down assay\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e pull-down analyses were performed using 50 \u0026micro;g of GST-Get3b and either 20 \u0026micro;g of Alb3C-His or 20 \u0026micro;g of Alb4C-His in a total volume of 120 \u0026micro;l of 1x PBS. Negative control reactions were conducted using GST instead of GST-Get3b or the His-tagged constructs alone. The samples were incubated for 10 min at room temperature. Subsequently, 100 \u0026micro;l of a 50% slurry of glutathione resin (equilibrated in 1x PBS) were added and incubated for 30 min at room temperature. The mixture was transferred to Wizard\u0026reg; Mini Columns (Promega) and centrifuged briefly. The column was washed with 5 ml of 1xPBS buffer using a syringe. After a brief centrifugation of the column the proteins were eluted with 30 \u0026micro;l of 10 mM reduced glutathione in 1xPBS buffer. The load as well as the eluted samples were analyzed by SDS-PAGE and proteins detected by Coomassie staining or Western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of split-ubiquitin plasmids and the split-ubiquitin yeast two-hybrid assays\u003c/h2\u003e \u003cp\u003eFor the split-ubiquitin yeast two-hybrid assay the coding sequence for mature full-length GET3b (aa 68\u0026ndash;411) was cloned into pADSL-Nx using the BamHI/SalI restriction sites. All other constructs have been described previously (Bals et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Pasch et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The split-ubiquitin assay was done according to (Pasch et al, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMS/MS analysis\u003c/h2\u003e \u003cp\u003eFor in-solution mass spectrometry, proteins were digested (Le\u0026oacute;n et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; ISD:SDC; AP\u0026amp;PT) and peptides were purified by C18 STAGE-TIPS (Rappsilber et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Peptides were analyzed using an ultra-HPLC Proxeon EASY-nLC 1000 system coupled online to Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Reversed-phase separation was performed using a 30 cm analytical column (100 \u0026micro;m diameter; DNU-MS (Novak) packed in-house with Reprosil-Pur 120 C18-AQ, 2.4 \u0026micro;m). Mobile-phase solvent A consisted of 0.1% formic acid and 4% acetonitrile in water, and mobile-phase solvent B consisted of 0.1% formic acid in 80% acetonitrile. The flow rate of the gradient was set to 200 nl/min. A 70-min gradient was used (0\u0026ndash;40% solvent B within 40 min, 40\u0026ndash;100% solvent B within 10 min, 100% solvent B for 10 min, 100\u0026ndash;0% solvent B within 5 min and 0% solvent B for 5 min).\u003c/p\u003e \u003cp\u003eData acquisition was performed with the ddMS2 method with the following configuration: For the MS scans, the scan range was set to 250\u0026ndash;2,000 m/z at a resolution of 70,000, and the automatic gain control (AGC) target was set to 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e. For the MS/MS scans, Top 13 ions were chosen, the resolution was set to 35,000, the AGC target was set to 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, the precursor isolation width was 2 Da and the maximum injection time was set to 80 ms. The HCD normalized collision energy was 27%. MaxQuant was used to analyze the LC-MS/MS data (ver 1.5.5.; Cox and Mann, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) which allowed qualitative and quantitative analysis. The UniProt reference \u003cem\u003eArabidopsis\u003c/em\u003e database (UP000006548) was used for the identification of proteins. Default settings for fixed modifications were used, Dynamic modifications were set: Oxidation for M and Deamidation for NQ. Contaminants were included for peptide detection of a minimum length of 6 amino acids. FDR threshold was set to 0.01. Perseus was used to transform the quantitative data and perform a statistical analysis (Tyanova and Cox, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The default settings for a Student\u0026rsquo;s t-test were utilized for the analysis. For this, independent triplicates corresponding to roughly 80 \u0026micro;g of plastid protein isolated from 8 pots of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e lawn from wild-type and \u003cem\u003eget3b\u003c/em\u003e plants were compared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAccession numbers\u003c/h2\u003e \u003cp\u003eThe following Arabidopsis genes were investigated in this study: AT3G10350 (GET3B), AT5G03940 (cpSRP54), AT1G24490 (ALB4/STIC1), and AT2G24020 (STIC2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eProteomic characterization of the\u003c/b\u003e \u003cb\u003eget3b\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor the GET3B locus, three T-DNA insertion lines are publicly available (Xing et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We decided to analyze the \u003cem\u003eget3b-2\u003c/em\u003e mutant (from here on \u003cem\u003eget3b\u003c/em\u003e) with an insertion in the fourth intron of the allele. The line was homozygous for the mutation and had no transcript nor protein detectable by RT-PCR or western blot, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D).\u003c/p\u003e \u003cp\u003eTo analyze the overall effect of the \u003cem\u003eget3b\u003c/em\u003e mutation on chloroplast function, we examined \u003cem\u003eget3b\u003c/em\u003e chloroplasts on a proteomic level. By using wild-type and \u003cem\u003eget3b\u003c/em\u003e plastids, a label-free technique could be employed in a quantitative manner as previously described (Vermeulen et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). For this, we isolated plastids of the respective lines from 21-day-old \u003cem\u003eA. thaliana\u003c/em\u003e seedlings and subjected these to LC-MS/MS.\u003c/p\u003e \u003cp\u003eThe examination revealed a total of 999 proteinaceous hits. After subtraction of contaminants, 203 were classified as significantly depleted and 105 as enriched in \u003cem\u003eget3b\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Intriguingly, not only nuclear encoded proteins, but also plastome encoded proteins were significantly affected. These covered a wide range of biological processes and were manually curated and categorized according to their cellular functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). The majority of the significantly depleted hits (33.9%) belonged to the category \u003cem\u003eother metabolic pathways\u003c/em\u003e, which included components of the AA synthesis machinery as well as the post-translational modification machinery. Apart from that, \u003cem\u003echaperone/assembly factors\u003c/em\u003e (10.3%), the translational machinery (9.8% \u003cem\u003eribosomes\u003c/em\u003e plus 5.9% \u003cem\u003etranscription/translation\u003c/em\u003e), the photosynthetic machinery (9.8% \u003cem\u003elight reaction\u003c/em\u003e plus 2.9% \u003cem\u003edark reaction\u003c/em\u003e) were mostly affected. Constituents of \u003cem\u003estarch synthesis\u003c/em\u003e (5.4%), the plastid \u003cem\u003eredox\u003c/em\u003e regulation and \u003cem\u003etranslocation\u003c/em\u003e (3.4% each), tRNA (2.9%), \u003cem\u003eproteases\u003c/em\u003e (2.4%), \u003cem\u003eFe-S cluster\u003c/em\u003e synthesis (1.4%), \u003cem\u003efatty acids\u003c/em\u003e and \u003cem\u003emembranes\u003c/em\u003e (0.9% each) were also depleted to a significant extent in \u003cem\u003eget3b\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Interestingly, one entire paralogous set of nuclear encoded proteins of the oxygen evolving complex (OEC; Allahverdiyeva et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) was reduced to a significant level. Furthermore, several components of the electron transport chain were also decreased. Additionally, soluble proteins involved in the Benson-Calvin cycle like several subunits of ribulose-1,5-bisphosphat-carboxylase/-oxygenase (RuBisCo) and their assembly factors were also negatively affected as well as STIC2, a factor involved in stromal targeting networks (B\u0026eacute;dard et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Strikingly, GET3B was detectable even though it was significantly depleted (Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), suggesting that the T-DNA insertion might lead to a knock-down rather than a knock-out of the respective gene.\u003c/p\u003e \u003cp\u003eThe majority of the significantly enriched hits were attributed to the \u003cem\u003elight reaction\u003c/em\u003e of photosynthesis (39%), followed \u003cem\u003eby other metabolic pathways\u003c/em\u003e (21.9%) and components of \u003cem\u003etranslocation\u003c/em\u003e machineries (12.4%). Additionally, \u003cem\u003estarch\u003c/em\u003e synthesis (8.5%), \u003cem\u003eproteases\u003c/em\u003e (3.8%), constituents involved in \u003cem\u003etranscription/translation\u003c/em\u003e (2.8%), \u003cem\u003echaperone/assembly factors, redox\u003c/em\u003e regulation, \u003cem\u003eFe-S\u003c/em\u003e cluster biogenesis and \u003cem\u003efatty acid\u003c/em\u003e synthesis (1.9% each) as well as elements involved in membrane modulation (0.9%) were elevated to a significant extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Strikingly, nearly all LHCPs of PSII and a few of PSI were enriched in \u003cem\u003eget3b\u003c/em\u003e, while the respective cpSRP targeting machinery was unaffected (Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe comparative proteomic data displayed that the loss or reduction of GET3B has a pervasive effect on the proteome of plastids. Interestingly, the photosynthetic apparatus seemed to be affected the most.\u003c/p\u003e \u003cp\u003e \u003cb\u003eget3b\u003c/b\u003e \u003cb\u003emutation affects de novo assembly of photosystem II\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe data from the comparative proteomics indicated that the light reaction as well as assembly factors of the photosynthetic machinery, like YCF3 and CRR6, were affected in \u003cem\u003eget3b\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G, supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This prompted us to examine a possible involvement of GET3B function in photosystem assembly. In order to address this, we employed PAM measurements during de-etiolation of \u003cem\u003eA. thaliana\u003c/em\u003e seedlings. These measurements exploit the fluorescence solely emitted by PS II, since PS I is a fluorescence quencher (Brettel, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). We utilized Fv/Fm values as a read out for PS II functionality. By employing this during de-etiolation we were able to assess the extent of de novo formation of PS II. For this, seedlings were germinated and cultivated \u003cem\u003ein vitro\u003c/em\u003e on Murashige Skoog (MS) medium (Murashige and Skoog, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) for 4 days in the dark and in the morning of the fifth day illuminated normally. The course of de-etiolation and concomitant de novo PS II assembly was encompassed by PAM measurements. This occurred 1, 7 and 24 hours after the initial illumination.\u003c/p\u003e \u003cp\u003eWhen cultivated without a carbon source, \u003cem\u003eget3b\u003c/em\u003e was significantly hampered in the assembly of PSII in comparison to wild-type after 7 and 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA; top panel). In the presence of a carbon source, this difference was only apparent after 24 hours and not to such a high degree of significance, indicating that PS II functionality was still hindered in the absence or reduction of GET3B when grown heterotrophically (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA; bottom panel).\u003c/p\u003e \u003cp\u003eAs a control we grew the same plates for five days without etiolation and analyzed these fluorometrically. When cultivated autotrophically, \u003cem\u003eget3b\u003c/em\u003e was still significantly obstructed in PS II assembly (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; top panel). Conversely, when grown in the presence of a carbon source, no difference was detectable with PAM measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; bottom panel).\u003c/p\u003e \u003cp\u003eBeing able to observe significant differences after five days of growth under normal irradiation, prompted us to analyze 4-day-old seedlings under regular illumination in the absence of sucrose. The corresponding PAM measurements mirrored the previous assessment and confirmed an impaired PSII assembly in \u003cem\u003eget3b\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Consistently, \u003cem\u003eget3b\u003c/em\u003e also exhibited a significant reduction in chlorophyll content (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTaken together these data indicated that, in \u003cem\u003eget3b\u003c/em\u003e mutants, not only the initial stages (up to 24 hours) of de novo assembly of PS II were affected but also the later stages (up to 5 days). Additionally, the reduced chlorophyll content of \u003cem\u003eget3b\u003c/em\u003e illustrated the obstruction of the photosynthetic machinery during early plant development.\u003c/p\u003e \u003cp\u003e \u003cb\u003eget3b\u003c/b\u003e \u003cb\u003emutation affects CO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eassimilation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe proteomic analysis also suggested that components of the dark reaction were affected by the \u003cem\u003eget3b\u003c/em\u003e mutation. Interestingly, not only three of four paralogous genes of the nuclear encoded small subunits but also the plastom encoded large subunit of RuBisCo were affected by the reduction or absence of GET3B. Additionally, RuBisCo assembly factors like RbcX, CPN60A1, CPN60B1 and CPN10-2 were significantly affected in \u003cem\u003eget3b\u003c/em\u003e (Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In total, these constitute six out of nine components necessary to synthesize RuBisCo \u003cem\u003ein vitro\u003c/em\u003e (Aigner et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In order to address this matter, we analyzed the CO\u003csub\u003e2\u003c/sub\u003e assimilation of \u003cem\u003eA. thaliana\u003c/em\u003e seedlings by Infra-red gas analyzer (IRGA; Toro et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The measurements revealed that \u003cem\u003eget3b\u003c/em\u003e assimilated significantly less CO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e). During the measurements we included a complementation line that was previously used (Xing et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The complementation line was able to rescue the \u003cem\u003eget3b\u003c/em\u003e assimilation phenotype and restore the levels above those of the wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis and the previous data support the results of the proteomic analysis, that GET3B is required for an efficient light as well as dark reaction of photosynthesis.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGET3B interacts genetically with components of the STIC pathway (ALB4 and STIC2)\u003c/h2\u003e \u003cp\u003ePreviously we had shown the negative -synergistic effect of combining the \u003cem\u003eget3b\u003c/em\u003e and \u003cem\u003esrp54\u003c/em\u003e alleles (Anderson et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A functional interaction between the two factors also became apparent from our proteomic analysis, in which \u003cem\u003eget3b\u003c/em\u003e displayed a significant enrichment of cpSRP substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e, supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This could be due to a form of compensation for the absence or reduction of GET3B function or the reduction of some stromal proteases which we could also observe in the proteomic data (Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Additionally, the significant depletion of STIC2 in \u003cem\u003eget3b\u003c/em\u003e prompted us to analyze the connection between the GET3B-, cpSRP- and the STIC- components.\u003c/p\u003e \u003cp\u003eHence, we utilized the existing single mutants \u003cem\u003esrp54\u003c/em\u003e (Amin et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), \u003cem\u003estic2\u003c/em\u003e (B\u0026eacute;dard et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003ealb4\u003c/em\u003e (Gerdes et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and \u003cem\u003eget3b\u003c/em\u003e, and double mutants \u003cem\u003esrp54 get3b\u003c/em\u003e (Anderson et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), \u003cem\u003ealb4 srp54\u003c/em\u003e, and \u003cem\u003ealb4 stic2\u003c/em\u003e (B\u0026eacute;dard et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and combined mutant alleles to create the double mutants \u003cem\u003ealb4 get3b\u003c/em\u003e and \u003cem\u003estic2 get3b\u003c/em\u003e, and triple mutants \u003cem\u003ealb4 stic2 get3b\u003c/em\u003e and \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003e). To assess the phenotypic consequences of these genetic combinations, we measured the rosette diameter as an indicator of overall plant fitness. Unlike the \u003cem\u003eget3b\u003c/em\u003e, \u003cem\u003ealb4\u003c/em\u003e, \u003cem\u003estic2\u003c/em\u003e single mutants and \u003cem\u003ealb4 stic2\u003c/em\u003e double mutant, the \u003cem\u003esrp54\u003c/em\u003e single mutant and \u003cem\u003ealb4 get3b\u003c/em\u003e, \u003cem\u003estic2 get3b\u003c/em\u003e double mutants displayed a significantly smaller rosette diameter compared to the wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-F, H, I, M). Strikingly, this phenotypical trait was further exacerbated in the \u003cem\u003esrp54 get3b\u003c/em\u003e double mutant and \u003cem\u003ealb4 stic2 get3b\u003c/em\u003e triple mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, J, M). These results illustrate an additive or synergistic interaction between GET3B and STIC components. Notably, the \u003cem\u003eget3b\u003c/em\u003e allele in a \u003cem\u003esrp54\u003c/em\u003e background showed a more severe phenotype than in an \u003cem\u003ealb4 stic2\u003c/em\u003e background, suggesting a stronger functional interdependence between GET3B and SRP54.\u003c/p\u003e \u003cp\u003eIn order to better understand this interdependence as well as the effect of the \u003cem\u003eget3b\u003c/em\u003e allele on plant fitness, we analyzed the \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e triple mutant. While \u003cem\u003ealb4 srp54\u003c/em\u003e plants were smaller than \u003cem\u003esrp54\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, M), the addition of the \u003cem\u003eget3b\u003c/em\u003e allele resulted in a further reduction in the measured rosette diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003eL, M). This demonstrated that the combination of the \u003cem\u003ealb4\u003c/em\u003e, \u003cem\u003esrp54\u003c/em\u003e and \u003cem\u003eget3b\u003c/em\u003e alleles resulted in an additive detrimental effect on plant fitness.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe ATPase function of GET3B is responsible for the growth phenotype\u003c/h2\u003e \u003cp\u003eIn order to investigate the restorative effects of GET3B expression in the \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e triple mutant, we complemented the triple mutant by transfecting the plants with a construct previously used (Anderson et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the T2 generation, 12 independent lines were identified carrying the \u003cem\u003eProUB10:GET3B-STREP\u003c/em\u003e transgene. Data are shown for 3 randomly chosen lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C, G). The rosette diameters of the three lines were significantly smaller (lines 2,3) as well as larger (line 1) than the \u003cem\u003ealb4 srp54\u003c/em\u003e double mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C, G). However, in all of the lines, rosette diameters were more than three times larger than the \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e triple mutant, signifying that the transgene was able to rescue the triple mutant phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003eTo determine if the molecular complementation was due to the targeting or holdase function of GET3B, we introduced \u003cem\u003eGET3B\u003c/em\u003e transgenes with D124N mutations into the \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e background (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). These have also been previously used (Anderson et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) to eliminate the ATPase activity of GET3B (Mateja et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009b\u003c/span\u003e). In the T2 generation 7 independent lines were identified that contained the \u003cem\u003eProUB10:GET3B (D124N)-STREP\u003c/em\u003e transgene. Data are shown for 4 randomly chosen lines. One of the lines (line2) displayed a significantly larger rosette diameter than the triple mutant but all of the lines were approximately three times smaller than the \u003cem\u003ealb4 srp54\u003c/em\u003e double mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-H). Overall, the transgene with a D124N mutation was unable to rescue the \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e phenotype, illustrating that the ATP hydrolysis activity is crucial for complementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eTo further investigate the functional consequences of the WT GET3B transgene and the ATP hydrolysis variant, we transfected these constructs into \u003cem\u003ealb4\u003c/em\u003e or \u003cem\u003estic2\u003c/em\u003e backgrounds. In the T2 generation, we observed distinct phenotypic differences between the untransfected and transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Transfection of the WT GET3B transgene into \u003cem\u003estic2\u003c/em\u003e backgrounds did not result in significant phenotypic differences compared to \u003cem\u003estic2\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D, G). In contrast, transfection of the ATP hydrolysis-deficient GET3B variant into the \u003cem\u003ealb4\u003c/em\u003e background led to a significantly smaller plant size in one of two randomly chosen lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, H). This effect was more pronounced when the transgene was transfected into a \u003cem\u003estic2\u003c/em\u003e background. Both of the two randomly chosen lines showed highly significant reductions in rosette diameter when compared to \u003cem\u003estic2\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, H).\u003c/p\u003e \u003cp\u003eThese findings show that the loss of ATP hydrolysis of GET3B in conjunction with impairments of the STIC pathway result in stunted growth. This clearly illustrates the importance of both functioning pathways for normal plant growth and chloroplast function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGET3B interacts physically with the C-termini of ALB3 and ALB4\u003c/h2\u003e \u003cp\u003eDue to the fact that the \u003cem\u003ealb3\u003c/em\u003e mutant does not survive beyond the seedling stage when germinated on soil (Sundberg et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), we did not include it in our preceding genetic analysis. Still we wanted to analyze the interplay of GET3B with ALB3 as well as ALB4 and verify if the latter two could function as the terminal stromal insertases of the pathway. We were interested in both homologues since these were suggested to engage with similar sets of interactors (Tr\u0026ouml;sch et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo investigate the interaction between GET3B and ALB3 or ALB4, we recombinantly expressed GET3B C-terminally fused to GST (GST-Get3b) as well as C-terminal fragments of ALB3 and ALB4 N-terminally fused to a His-tag (Alb3C-His and Alb4C-His; Ackermann et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and performed pulldown experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). We mixed GST-Get3b with Alb3-His as well as GST-Get3b with Alb4C-His and incubated them with Glutathione Sepharose. In order to check for unspecific interactions, Alb3C- and Alb4C-His were incubated with GST and Glutathione Sepharose resin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We observed residual amounts of both Alb3C- and Alb4C-His being pulled down by Glutathione Sepharose and the GST tag, indicating a non-specific interaction with the resin. However, when GST-Get3b was included in the pulldown experiment, we observed a clear enrichment of both Alb3C- and Alb4C-His, suggesting a specific interaction between GET3B and the C-terminus of ALB3 and ALB4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn order to corroborate the \u003cem\u003ein vitro\u003c/em\u003e findings we employed a yeast two-hybrid split ubiquitin assay to further investigate the interaction between GET3B and ALB3 as well as ALB4. We transfected yeast cells with constructs expressing full length ALB3 or ALB4 N-terminally fused to the C-terminus of ubiquitin (Alb3-Cub and Alb4-Cub), along with GET3B fused to the N-terminus of ubiquitin (NubG-Get3b) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). As controls, we transfected cells with Alg5 fused to the N-terminus of ubiquitin, both with and without the mutation allowing spontaneous reassembly of ubiquitin (Alg5-Nubl and Alg5-NubG, respectively). As additional controls, we tested the C-terminus of ubiquitin N-terminally fused to SEC1 and ALG5 (Sec1-Cub and Alg5-Cub) together with NubG-Get3b (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eUpon growth on the corresponding dropout medium, we observed robust cell growth in combination with NubG-Get3b and Alb3-Cub as well as Alb4-Cub fusion constructs, indicating a positive interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D second column). In contrast, cells co-transfected with the control protein, Alg5-NubG as well as Sec1-Cub did not exhibit significant growth under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eE second column). As positive controls, cells expressing Alg5-NubG grew on all media, confirming the functionality of the assay.\u003c/p\u003e \u003cp\u003eThe combination of the pulldown experiments as well as the yeast two-hybrid split ubiquitin experiment indicated a physical interaction between GET3B and the C-terminus of ALB3 and ALB4. These results together with previous ones (Anderson et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) support the notion that GET3B selectively binds clients and interacts with insertases, potentially facilitating the membrane integration of proteins within chloroplasts.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe findings of this study illuminate the critical role of \u003cem\u003eAt\u003c/em\u003eGET3B in chloroplast function. We provide new mechanistic insights of its function during photosynthesis and chloroplast biogenesis in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e.\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eImpact of GET3B on chloroplast proteome, PS II assembly and carbon fixation\u003c/h2\u003e \u003cp\u003eThe proteomic analysis provided a comprehensive overview of the downstream effects caused by the absence or reduction of GET3B. This was crucial in identifying the specific proteins and pathways most affected by the \u003cem\u003eget3b\u003c/em\u003e mutation, shedding light on the multifaceted role of GET3B in chloroplast biogenesis and function. One of the most striking findings from the proteomic analysis was the global effect on the abundance of plastidic proteins in the \u003cem\u003eget3b\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These encompass a wide functional range, suggesting that GET3B has a broad impact on chloroplast proteostasis.\u003c/p\u003e \u003cp\u003eThe entire photosynthetic apparatus of higher plats contains 16 single pass IMPs (Shi and Schr\u0026ouml;der, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2004\u003c/span\u003e): PS II contains 12 of which 7 have a TA topology (Wei et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) PS I comprises two of which none have a TA topology (Pan et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and the ATP synthase encompasses two of which one shares a TA topology (Hahn et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Taking the postulated function of GET3B into account as the central element of the stromal TA targeting machinery one would expect the corresponding substrate to be reduced in a genetic background of a knock-out or knock-down. Remarkably, the only single pass IMP that was significantly depleted in \u003cem\u003eget3b\u003c/em\u003e was PsbR (Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Together with PsbO, PsbP and PsbQ, PsbR forms the OEC and represents its only IMP (Mishra and Ghanotakis, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). To date structural analyses of PS II of higher plants were unable to resolve the location and topology of PsbR (Van Bezouwen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, previous biochemical analyses corroborated that the majority of the elongated N-terminus is oriented to the thylakoidal lumen (Ljungberg et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Ljungberg et al., 1986). If this topology is correct, then no bona fide photosynthetic TA protein was significantly depleted by the absence or reduction of GET3B. This implies that either GET3B is not involved in the targeting of TA proteins or another factor can compensate for its absence (see next section: genetic and physical interactions with STIC component and SRP54).\u003c/p\u003e \u003cp\u003eThe significant reduction of Fv/Fm values during the de novo assembly of PS II highlight the important role of GET3B in early chloroplast biogenesis. The partial restoration of emitted fluorescence and concomitant PS II functionality in the presence of an external carbon source suggests that GET3B may play a crucial role under autotrophic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). The observed reduction of Fv/Fm values during de-etiolation could either arise from impediments in OEC assembly/function or the depletion of key photosynthetic assembly factors (Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). It is difficult to dissect whether this reduction actually results from an incomplete assembly of PS II or solely an impeded OEC of PS II. Further analyses are necessary to clarify this.\u003c/p\u003e \u003cp\u003eComponents of chlorophyll biogenesis pathways such as CHL synthase, magnesium chelatase and several assembly factors as well as subunits of RuBisCo were significantly depleted in \u003cem\u003eget3b\u003c/em\u003e (Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This directly correlates with the reduced chlorophyll content of young \u003cem\u003eget3b\u003c/em\u003e seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) as well as the reduced carbon assimilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e), respectively. These observations further support the conclusion that GET3B is integral to the proper formation and function of the entire photosynthetic apparatus. However, at this stage it is hard to dissect if these are due to the direct function of GET3B or secondary pleiotropic effects.\u003c/p\u003e \u003cp\u003eThe proteomic analysis provided critical insight into the molecular underpinnings of the \u003cem\u003eget3b\u003c/em\u003e mutation. GET3B appears to be vital for the maintenance of a wide range of chloroplast proteins, particularly those involved in photosynthesis. The broad impact of GET3B on chloroplast proteome underscores its central role in chloroplast biogenesis and function, with potential implications for understanding how chloroplast proteostasis is maintained under various physiological conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGenetic and physical interactions with STIC components and SRP54\u003c/h2\u003e \u003cp\u003eThe negative synergism derived from combining the \u003cem\u003eget3b\u003c/em\u003e allele with \u003cem\u003esrp54\u003c/em\u003e, \u003cem\u003ealb4\u003c/em\u003e and \u003cem\u003estic2\u003c/em\u003e implies an orchestrated network between GET3B and these pathway components probably by ensuring the proper integration and assembly of chloroplast proteins.\u003c/p\u003e \u003cp\u003eIn particular, the \u003cem\u003esrp54 get3b\u003c/em\u003e double mutant displayed a more severe phenotype than either single mutant by itself (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003e), signifying that the \u003cem\u003esrp54\u003c/em\u003e mutation acts as a potentiator of the \u003cem\u003eget3b\u003c/em\u003e effect or vice versa. This suggests that SRP54 and GET3B either may work together in a single pathway or may work separately in parallel pathways that converge in similar chloroplastidic processes. Interestingly, SRP54 binds solely to the third TMD of LHCPs (High et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). This TMD is the most hydrophobic of all three and fusing it alone to a soluble protein will translocate the fusion protein into the thylakoid membrane (Kohorn and Tobin, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Since both SRP54 and GET3B are capable of binding single TMDs, it might be the case that these two proteins are able to partially compensate for the absence of the other, thereby maintaining stromal IMP trafficking when other targeting networks are not fully functional.\u003c/p\u003e \u003cp\u003eThe incapacity of the ATPase-deficient variant to rescue the growth phenotype in the \u003cem\u003ealb4 srp54 get3b\u003c/em\u003e triple mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003e) underscores that it is the targeting function of GET3B rather than the potential holdase function which enables its participation in chloroplastidic proteostasis. The observed growth defects of plants expressing the ATPase-defiant variant in \u003cem\u003ealb4\u003c/em\u003e and \u003cem\u003estic2\u003c/em\u003e backgrounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003e) further confirm that operative ATPase activity is essential for a fully functioning GET3B in planta.\u003c/p\u003e \u003cp\u003eThe physical interaction between GET3B and the C-terminal regions of ALB3 and ALB4, as demonstrated by pulldown experiments and yeast two-hybrid assays, provides a mechanistic insight into how GET3B may facilitate the insertion of proteins into the thylakoid membrane. Interestingly, irrespective of \u003cem\u003ein vitro\u003c/em\u003e or \u003cem\u003ein vivo\u003c/em\u003e, both the stromal exposed C-terminal regions or full length ALB3 and ALB4 interacted with GET3B to a fairly similar degree (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003e), indicating less selectivity for the insertases than the cpSRP pathway. The C-terminal regions of ALB3 and ALB4 share two conserved motifs: motif I and motif III, which are not utilized by ALB3 for the interaction with SRP43 (Falk et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In turn, SRP43 interacts with motifs II and IV of ALB3 which helps it discriminate between the two insertases (Falk et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; D\u0026uuml;nschede et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Recently, a direct physical interaction between STIC2 and motif III of ALB3 and ALB4 was demonstrated (Stolle et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The plastidic ribosome on the other hand cooperatively binds motif III and IV of ALB3 (Ackermann et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In future studies, it would be interesting to characterize the GET3B binding site within the C-terminal regions of the Alb proteins and to investigate whether GET3B and STIC2 may engage in competitive or cooperative binding.\u003c/p\u003e \u003cp\u003eIn summary, the results of this study highlight the role of GET3B in chloroplast biogenesis and function. GET3B appears to be involved in the proper assembly and function of photosynthetic complexes but also for proteostasis in a broader sense within the chloroplast. Its genetic interactions with STIC components and SRP54, as well as its dependence on ATPase activity, suggests a critical role in the integration of membrane proteins. Further studies are necessary to explore the precise mechanistic details of GET3B\u003cb\u003e\u0026rsquo;\u003c/b\u003es interactions with the other targeting components and how they contribute to the overall regulation of chloroplast biogenesis and function.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like thank Sarah Friedrich for plant photographs and figure preparation as well as Fabian Fuchs and Silke Funke for experimental support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Compliance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript does not include data or description of human or animal patients\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Deutsche Forschungsgemeinschaft (DFG SCHL 585-10) and the University of Wisconsin-Madison Office of the Vice Chancellor for Research and Graduate Education.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: [Uwe Bodensohn, Danja Schuenemann, Donna Fernandez and Enrico Schleiff]; Genetic analyses: [Donna Fernandez]; Interaction analyses: [Beatrix D\u0026uuml;nschede, Chiara Kuhlmann]; IRGA measurements: [Khushbu Kumari]; LC-MS/MS [Roman Ladig]; Physiological experiments: [Uwe Bodensohn]; Writing \u0026ndash; original draft preparation: [Uwe Bodensohn]; Writing \u0026ndash; review and editing: [Uwe Bodensohn, Danja Schuenemann, Donna Fernandez]; Funding acquisition: [Enrico Schleiff, Donna Fernandez]; Supervision: [Uwe Bodensohn, Danja Schuenemann, Donna Fernandez, Christopher Grefen, Enrico Schleiff] \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eAckermann, B., D\u0026uuml;nschede, B., Pietzenuk, B., Justesen, B.H., Kr\u0026auml;mer, U., Hofmann, E., G\u0026uuml;nther Pomorski, T., and Sch\u0026uuml;nemann, D.\u003c/strong\u003e (2021). 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A. \u003cstrong\u003e114\u003c/strong\u003e: E1544\u0026ndash;E1553.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eYu, B., Gruber, M., Khachatourians, G.G., Zhou, R., Epp, D.J., Hegedus, D.D., Parkin, I.A.P., Welsch, R., and Hannoufa, A.\u003c/strong\u003e (2012). Arabidopsis cpSRP54 regulates carotenoid accumulation in Arabidopsis and Brassica napus. J. Exp. Bot. \u003cstrong\u003e63\u003c/strong\u003e: 5189\u0026ndash;5202.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eZiehe, D., D\u0026uuml;nschede, B., and Sch\u0026uuml;nemann, D.\u003c/strong\u003e (2018). Molecular mechanism of SRP-dependent light-harvesting protein transport to the thylakoid membrane in plants. Photosynth. Res. \u003cstrong\u003e138\u003c/strong\u003e: 303\u0026ndash;313.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ALB3, ALB4, cpSRP54, GET3B, STIC1, STIC2","lastPublishedDoi":"10.21203/rs.3.rs-6212458/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6212458/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProtein targeting and insertion into membranes are essential for cellular organization and organelle function. The Guided Entry of Tail-anchored (GET) pathway facilitates the post-translational targeting and insertion of tail-anchored (TA) membrane proteins. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e has four GET3 homologues, including \u003cem\u003eAt\u003c/em\u003eGET3B and \u003cem\u003eAt\u003c/em\u003eGET3D localized to chloroplasts. These photosynthetic organelles possess complex membrane systems, and the mechanisms underlying their protein targeting and membrane biogenesis are not fully understood. This study conducted a comprehensive proteomic analysis of \u003cem\u003eget3b\u003c/em\u003e mutant plastids, which displayed significant alterations. Fluorometric based complex assembly as well as CO\u003csub\u003e2\u003c/sub\u003e assimilation analyses confirmed that disruption of GET3B function displayed a significant impact on photosystem II assembly as well as carbon fixation, respectively, indicating a functional role in chloroplast biogenesis. Additionally, genetic interactions were found between GET3B and the two component STIC system, which cooperates with the cpSRP pathway and is involved in the co-translational sorting of thylakoid proteins. Further, physical interactions were observed between GET3B and the C-terminus of ALB3 and ALB4 \u003cem\u003ein vitro\u003c/em\u003e and the full length proteins \u003cem\u003ein vivo\u003c/em\u003e, indicating a role of GET3B in protein targeting and membrane integration within chloroplasts. These findings enhance our understanding of GET3B\u0026rsquo;s involvement in stromal protein targeting and thylakoidal biogenesis.\u003c/p\u003e","manuscriptTitle":"GET3B is involved in chloroplast biogenesis and interacts with the thylakoidal ALB3 and ALB4 insertases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 14:05:32","doi":"10.21203/rs.3.rs-6212458/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2025-04-10T18:18:02+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-21T00:35:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-20T21:55:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T13:07:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2025-03-17T13:49:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6bd1a0a8-5fd7-4bae-91e7-e9f2f8c6579a","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-05T16:03:10+00:00","versionOfRecord":{"articleIdentity":"rs-6212458","link":"https://doi.org/10.1007/s00299-025-03500-2","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2025-04-29 15:57:29","publishedOnDateReadable":"April 29th, 2025"},"versionCreatedAt":"2025-03-31 14:05:32","video":"","vorDoi":"10.1007/s00299-025-03500-2","vorDoiUrl":"https://doi.org/10.1007/s00299-025-03500-2","workflowStages":[]},"version":"v1","identity":"rs-6212458","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6212458","identity":"rs-6212458","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}