Differential Involvement of Arabidopsis β’-COP Isoforms in Plant Development

Coat protein I (COPI) is necessary for intra-Golgi transport and retrograde transport from the Golgi apparatus back to the endoplasmic reticulum. The key component of the COPI coat is the coatomer complex, which is composed of seven subunits (α/β/β’/γ/δ/ε/ζ) and is recruited en bloc from the cytosol onto Golgi membranes. In mammals and yeast, α- and β’-COP WD40 domains mediate cargo-selective interactions with dilysine motifs present in canonical cargoes of COPI vesicles. In contrast to mammals and yeast, three isoforms of β’-COP (β’1-3-COP) have been identified in Arabidopsis. To understand the role of Arabidopsis β’-COP isoforms in plant biology, we have identified and characterized loss-of-function mutants of the three isoforms, and double mutants were also generated. We have found that the trafficking of a canonical dilysine cargo (the p24 family protein p24δ5) is affected in β’-COP double mutants. By western blot analysis, it is also shown that protein levels of α-COP are reduced in the β’-COP double mutants. Although none of the single mutants showed an obvious growth defect, double mutants showed different growth phenotypes. The double mutant analysis suggests that, under standard growth conditions, β’1-COP can compensate for the loss of both β’2-COP and β’3-COP and may have a prominent role during seedling development.


Introduction
Coat Protein I (COPI)-coated vesicles are involved in transport between Golgi cisternae and in retrograde transport from the Golgi apparatus back to the endoplasmic reticulum (ER) [1]. In mammalian cells, COPI proteins have been recently proposed as also playing a role in the last step of anterograde ER-Golgi transport [2,3].
The COPI coat is based on a cytosolic complex (coatomer), containing seven equimolar subunits (α-, β-, β'-, γ-, δ-, εand ζ-COP), which interacts with Golgi membranes via the GTPase ADP-ribosylation factor 1 (ARF1). Cytosolic (GDP-bound) ARF1 first interacts with dimers of p24 family proteins, but following GTP/GDP exchange, ARF1-GTP dissociates from p24 proteins and inserts into Golgi membranes. Coatomer can then interact both with ARF1-GTP and with sorting signals (i.e., dilysine motifs) in the cytosolic domain of p24 family proteins and other COPI cargo proteins. Coatomer polymerization induces COPI vesicle formation, whereas COPI uncoating requires GTP hydrolysis in ARF1 [1,4,5]. In contrast to clathrin and COPII coats, where the inner layer (involved in cargo recognition) and the outer layer (involved in membrane deformation) are recruited sequentially, COPI coatomer is recruited en bloc from the cytosol onto Golgi membranes [5,6]. However, biochemical studies have shown that coatomer is composed of two subcomplexes, the B-subcomplex, containing the α-, β'and ε-COP subunits, and the F-subcomplex, containing the β-, γ-, δand ζ-COP subunits. The F-subcomplex is structurally very similar affect tolerance to salt stress, in particular to chloride ions, possibly due to mislocalization or reduced activity of chloride channels/transporters [32,33]. Here we have used a lossof-function approach to analyze the possible function of the three paralogs of the β'-COP subunit in Arabidopsis.

Plant Material and Stress Treatments
Arabidopsis thaliana (ecotype Col-0) was used as wild type. The loss-of-function mutants β'1-cop-1 (SALK_206753), β'1-cop-2 (WiscDsLoxHs036_02G), β'2-cop-1 (SALK_056771), β'3-cop-1 (SALK_004817), β'3-cop-2 (SALK_206870) and β'3-cop-3 (SALK_096549) were from the Salk Institute Genomic Analysis Laboratory and obtained from the Nottingham Arabidopsis Stock Centre. In β'1-cop-1 and β'3-cop-2 mutants, next-generation sequencing detected only one T-DNA insertion. Mutant lines were characterized by PCR (Supplementary  Table S1). A. thaliana plants were grown in growth chambers under a 16-h-light:8-h-dark regime and 75% relative humidity at 21 • C. To study whether salt tolerance was affected in the β'-COP double mutants, seeds of wild type (Col-0) and mutants were sown on Murashige and Skoog (MS) plates containing 135-150 mM NaCl. Plates were transferred to a controlled growth chamber after cold treatment in the dark for three days at 4 • C. After 12 days, the rates of cotyledon greening were scored. To study KCl (100-110 mM) tolerance, the same protocol was used. Seeds harvested from Col-0 and mutant plants grown under the same conditions and at the same time were used.

Reverse Transcription PCR (RT-PCR)
Total RNA was extracted from seedlings using a NucleoSpin RNA plant kit (Macherey-Nagel, Düren, Germany), and 3 µg of the RNA solution were reverse transcribed using the maxima first strand cDNA synthesis kit for quantitative RT-PCR (Fermentas, Burlington, ON, Canada), according to the manufacturer's instructions. Semi-quantitative PCRs (sqPCRs) were performed on a cDNA template using the PCR Master kit (Emeral-dAmp Max-2X Premix) (TaKaRa Bio, Shiga, Japan). The sequences of the primers used for PCR amplifications are included in Supplementary Table S2. Quantitative PCR (qPCR) was performed by using a StepOne Plus machine (Applied Biosystems, California, CA, USA) with SYBR Premix Ex Taq TM (Tli RNaseH Plus) (TaKaRa Bio), according to the manufacturer's protocol. Each reaction was performed in triplicate with 100 ng of the firststrand cDNA and 0.3 mM of primers for all the genes and 0.9 mM for SEC31A in a total volume of 20 µL. The specificity of the PCR amplification was confirmed with a heat dissociation curve (from 60 to 95 • C). Relative mRNA abundance was calculated using the comparative Ct method, according to Pfaffl [34]. Primers used for qPCR are listed in Supplementary Table S3.

Transgenic RFP-p24δ5 Plants
Transgenic plants were generated by transformation of Col-0, β'1β'3-cop-1 and β'2β'3cop-2 plants with the RFP-p24δ5 construct via Agrobacterium using the floral dip method, according to standard procedures [35]. The RFP-p24δ5 construct has been previously described and encodes a RFP fusion protein with a mRFP located at the N-terminus of the protein (right after the signal sequence and before the N-terminus of the mature p24δ5 protein) under the control of the 35S promoter [30,36]. The fluorescence of the mRFP used has been shown to be highly stable at the acidic pH of the vacuole lumen [36,37]. T1 plants were analyzed by confocal microscopy.

Confocal Microscopy
Imaging was performed using an Olympus FV1000 confocal microscope with a 60× water lens. A fluorescence signal for RFP (543 nm/593-636 nm) was detected. Sequential scanning was used to avoid any interference between fluorescence channels. Post-acquisition image processing was performed using the FV10-ASW4.2 Viewer ® .
To confirm CRISPR-Cas9-mediated editing of the target gen, young leaves' genomic DNA was obtained following the protocol described by Edwards [41] and the PCRs were performed using specific primers (Supplementary Table S4). For Sanger sequencing, obtained PCR products were purified and sequenced by Macrogen Co. (Madrid, Spain). The sequencing was carried out using a specific primer (Supplementary Table S4). Chromatograms from sequencing results were analyzed by Synthego "ICE CRISPR analysis tool" (https://ice.synthego.com/#, accessed on 13 January 2022).

Protein Extracts, SDS-PAGE and Immunoblotting
Cotyledons of seven-day-old wild type plants and loss-of-function mutants were ground in homogenization buffer (HB, 0.3 M sucrose; 1 mM EDTA; 20 mM KCl; 20 mM HEPES pH 7.5) supplemented with 1 mM DTT and a Protease Inhibitor Cocktail (Sigma-Aldrich Co., St. Louis, MO, USA), using a mortar and pestle. The homogenate was centrifuged two times for 5 min at 1200× g and 4 • C, and the post nuclear supernatant (PNS) was collected. Then, the PNS was centrifuged for 10 min at 1,000,000× g and 4 • C, and the supernatant was collected as a cytosolic extract.
Protein quantitation was performed by using the Bradford assay (Bio-Rad Laboratories GmbH, Munich, Germany). Protein extracts were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were then transferred to nitrocellulose membranes (Schleicher and Schuell, Maidstone, UK). Membranes were stained with Ponceau S solution (Sigma) before incubation with primary antibodies against COPI subunits and peroxidaselabeled secondary antibodies. The luminescent signal was developed using the SuperSignal West Pico chemiluminescent substrate (Pierce-Thermo Scientific, Rockford, IL, USA). Polyclonal antibodies against mammalian β -COP (C1PL) and α-COP were kindly provided by Dr F. Wieland (Biochemie-Zentrum, Heidelberg, Germany). Immunoblots were analyzed using the ChemiDoc XRS + imaging system (Bio-Rad, California, CA, USA). Immunoblots in the linear range of detection were quantified using Quantity One software (Bio-Rad Laboratories), with the Ponceau stain protein as a loading control.

Statistical Analysis
Differences in stress responses, protein levels in western blotting analysis and mRNA levels in RT-sqPCR were tested using a two-sample t-test with unequal variances (Microsoft Excel 2013) among all the β'-COP mutants compared to Col-0.
To investigate the relative expression of β'-COP genes, we used the publicly available expression database GENEVESTIGATOR [42,43] and ePlant-BAR [44]. As shown in Figure 1A, the three genes show high expression levels throughout plant development. The main difference between the three isoforms is their expression during seed development. As it is shown in Figure 1B, β'1-COP had the highest expression levels, up to four times higher than β'2-COP and six times higher than β'3-COP, in the last stages of seed development. This suggests that β'1-COP might exert a function in seed development. In contrast, β'3-COP showed the lowest expression levels at these stages.  [44][45][46]. Gene expression data generated by the Affymetrix ATH1 array are normalized by the GCOS method, TGT value of 100. Tissues were sampled in triplicate. The legend at the left presents relative expression levels coded by colours (blue = low, red = high).
None of the β'2-cop and β'3-cop mutants showed any obvious phenotypic alteration under standard growth conditions when compared to wild type plants, and only β'1-cop-1 mutant showed slightly reduced plant height (Supplementary Figure S2C).

(C) β'2β'3-cop-1, β'2β'3-cop-2 and β'2β'3-cop-3 mutants show
upregulation of the COPII subunit SEC31A gene. Expression of SEC31A and SEC31B was analyzed by RT-qPCR. Total RNA was extracted from 7-day-old seedlings of the mutants and wild type (Col-0). The mRNA was analyzed by RT-qPCR with specific primers and normalized to UBQ10 expression (Supplementary Tables S1-S3). Results are from three biological samples and three technical replicates. mRNA levels are expressed as relative expression levels and represent fold changes of mutant over wild type. Values represent mean ± s.e.m. of the three biological samples.

Loss of Two β'-COP Isoforms Causes a Reduction in the Protein Levels of α-COP
Next, we monitored β'-COP protein depletion in the mutants by using an antibody against mammalian β'-COP [48], since there are no Arabidopsis β'-COP antibodies available. As shown in Figure 5A, the β'-COP antibody recognized a clear band of approximately 100 kDa, corresponding to the molecular weight of β'-COP, in wild type (Col-0), β'1-cop-1, β'3-cop-1 and β'3-cop-2 mutants, while in β'2-cop-1 mutant, only a faint band was detected. The β'-COP antibody also recognized a clear band of approximately 100 kDa in β'1β'3-cop and a faint band in β'2β'3-cop ( Figure 5B). All these results suggest that mammalian β'-COP antibody has a higher affinity for β'2-COP than for the other isoforms.  [48,49]. β'1-COP antibodies were raised against the first 12 amino acids of cow β'1-COP. Cow β'-COP and Arabidopsis β'1-COP, β'2-COP and β'3-COP share 10, 11 and 10 amino acids, respectively. The β'-COP antibody detected a clear band of approximately 100 kDa, corresponding to the molecular weight of β'-COP, in wild type (Col-0), β'1-cop-1, β'3-cop-1 and β'3cop-2, and only a faint band in β'2-cop-1, suggesting that mammalian β'-COP antibody has higher affinity for β'2-COP. The different affinity for β'2-COP could be due to the sixth N-terminal amino acid of β'2-COP that is the same in of cow β'-COP and not in β'1-COP and β'3-COP. Alternatively, different splicing forms involved or postranslational modifications at the N-terminal might decrease the affinity of the antibody. α-COP antibodies have been previously shown to recognize both α1-COP and α2-COP isoforms and detected a band of approximately 130 kDa corresponding to the molecular weight of α-COP [30]. (B) Western blot analysis of cytosolic protein extracts from cotyledon of 7-day-old seedlings of wild type, β'1β'3-cop-1 and β'2β'3-cop-2 using mammalian β'-COP and α-COP N-terminal peptide antibodies. The β'-COP antibody recognized a clear band of approximately 100 kDa in β'1β'3-cop-1 and a faint band in β'2β'3-cop-2, suggesting again that mammalian β'-COP antibody has higher affinity for β'2-COP. Bottom panel shows the relative α-COP protein levels quantified of three biological samples. In (A,B), 12 µg of total protein was loaded in each lane. Ponceau protein stain was used as a loading control. (C) Relative expression levels of α-COP genes. Total RNA was isolated from 7-day-old cotyledon seedlings of wild type, β'1β'3-cop-1 and β'2β'3cop-2 mutants. RT-sqPCR analysis was performed with the primers listed in Supplementary Table S2. ACT7 was used as a control. Values represent mean ± s.e.m. of the three biological samples and were normalized against the band intensity in wild type that was considered to be 100%. Statistical significance: ns, not significant; * p < 0.05; ** p < 0.01.
In yeast, β'-COP depletion was shown to affect the levels of other COPI subunits, such as α-COP [11]. Therefore, we tested the effect of β'-COP depletion by Western blot analysis using an antibody against mammalian α-COP [49], that has been shown previously to recognize both isoforms of the Arabidopsis α-COP subunit [30]. Using this α-COP antibody we found that the levels of α-COP in the single β'-cop mutants were not affected ( Figure 5A). However, β'1β'3-cop-1 and β'2β'3-cop-2 double mutants showed lower levels of α-COP ( Figure 5B).

Loss of Two β'-COP Isoforms Causes Impaired Trafficking of p24δ5, a COPI Dilysine Cargo
β'-COP has been shown to bind to dilysine motifs, which are present in canonical COPI cargoes. One of these cargoes is p24δ5, a protein of the p24 family, which has been previously shown to localize to the ER due to COPI-dependent Golgi-to-ER transport based on a dilysine motif at its C-terminal tail [36,50]. Therefore, we investigated whether trafficking of RFP-p24δ5 was affected in β'-COP double mutants. As shown in Figure 6, RFP-p24δ5 localized to the ER in wild type transgenic plants. In contrast, in both β'1β'3-cop and in β'2β'3-cop mutants RFP-p24δ5 showed a predominant localization to the vacuole lumen, with some partial ER localization. This is consistent with impaired retrograde trafficking of p24δ5 from the Golgi back to the ER in the mutants and with previous results showing that transport to the vacuole may be a default pathway for membrane proteins in the plant secretory pathway [36]. This result is also consistent with the role of β'-COP in trafficking of dilysine cargoes. Confocal laser scanning microscopy of epidermal cells of 4.5-day-old cotyledons. All images shown were acquired using comparable photomultiplier gain and offset settings. RFP-p24δ5 mainly localized to the ER network in wild type plants (Col-0) (A,B) (see a z-stack projection in (B)). In contrast, it mainly localized to the vacuole lumen in β'1β'3-cop-1 (C,D) and β'2β'3-cop-2 (E,F) double mutants, although a partial ER localization was also found (C,E). Scale bars, 10 µm.

Discussion
Over the last years, several studies have been performed to elucidate putative specific functions of different COPI subunits in mammals. Particularly, a paralog-specific role has been proposed for the γand ζ-COP subunits, since these are the only COPI subunits codified by two different genes in mammals [4]. Proteomic studies of COPI vesicles generated in vitro with different γand ζ-COP isoforms, using HeLa cells as donor membranes, were not able to reveal a differential protein composition, arguing against selective cargo content [23]. However, it has been recently shown that γ1-COP and γ2-COP isoforms are differentially expressed during the neuronal differentiation of mouse pluripotent cells and, although they are functionally redundant to a large extent, γ1-COP specifically promotes neurite outgrowth [25].
Despite the fact that most COPI genes (including α-, β-, β'-, εand ζ-COP) have different paralogs in Arabidopsis, it is not yet known whether different COPI subunit isoforms are functionally redundant or may have specific functions, tissue, or development specificity, or perhaps bind different cargo proteins. Interestingly, and in contrast to mammals, morphologically different COPI vesicles have been described in plants [26], which might be formed by different COPI subunit isoforms, although this hypothesis still needs to be demonstrated.
In this work, we have used a loss-of-function approach to analyze the function of the three Arabidopsis β'-COP isoforms. To this end, we characterized single and double β'-COP mutants. Under standard growth conditions, none of the single β'-COP mutants displayed severe developmental defects, which was likely caused by at least partial functional redundancy among Arabidopsis β'-COP genes. β'-COP double mutant analysis under standard growth conditions suggests that β'3-COP cannot compensate for the simultaneous loss of β'1-COP and β'2-COP. Similarly, β'2-COP cannot compensate for the simultaneous loss of β'1-COP and β'3-COP, as the β'1β'3-cop double mutant failed to develop beyond the seedling stage. However, β'2β'3-cop double mutants had no major phenotypic alterations, indicating that β'1-COP does seem to compensate for the simultaneous lack of β'2-COP and β'3-COP. The results of double mutant analysis appear to correlate with the seed development expression patterns of β'1-COP and suggest a role of β'1-COP during seed development that may affect seedling growth.
Coatomer is made of seven equimolar COPI subunits and is recruited en bloc from the cytosol onto Golgi membranes. However, it is not clear how the levels of the different COPI subunits are regulated and how the absence of any of the subunits affects the structure of the complex and the stability of the other subunits. In this work, we found that the protein levels of another COPI subunit, α-COP, was not affected in the single β'-COP mutants. However, β'-COP double mutants showed a dramatic decrease in the levels of α-COP. This is consistent with the known interaction between αand β'-COP subunits in the B-subcomplex, and suggests that the α-COP subunit is destabilized in the absence of β'-COP. Strikingly, the sec27-1 yeast β'-COP mutant (harboring a point mutation in the carboxy-terminal region) also showed a reduction in the levels of α-COP [11]. This was proposed to be due to a local instability of the α-solenoid structure in β'-COP which would affect its interaction with α-COP. Strikingly, β'1β'3-cop-1, but not β'2β'3-cop-2, have also lower α1/α2-COP mRNA levels than wild-type, which may also contribute to the decrease in α-COP protein levels in this mutant. As α1-COP mRNA levels were more affected than α2-COP mRNA levels in β'1β'3-cop-1, it would be interesting to test in the future whether the isoform α1-COP is a specific partner of β'1-COP. Further experiments should be performed to clarify these issues.
The β'-COP subunit has been shown to play a role in binding to dilysine motifs in canonical COPI cargo proteins. Therefore, we hypothesized that loss of β'-COP may affect trafficking of dilysine cargo proteins, as observed in the sec27-1 yeast β'-COP mutant [11]. Indeed, we have found that the two β'1β'3-cop and β'2β'3-cop double mutants showed a mislocalization of p24δ5, which contains a cytosolic C-terminal dilysine motif, from the ER to the vacuole. This may be due to impaired COPI-dependent Golgi-to-ER transport of p24δ5 (Figure 7), which is mediated by its dilysine motif [36]. Indeed, we have shown previously that p24δ5 mutants lacking the dilysine motif were transported along the secretory pathway to the prevacuolar compartment and the vacuole, although a significant fraction was also found at the plasma membrane [36]. This suggests that transport to the vacuole is an alternate default pathway for membrane proteins in the secretory pathway. Therefore, both the absence of the dilysine motif or impaired COPI function have the same trafficking defect in p24δ5, a canonical COPI cargo. The sequences of Arabidopsis β'-COP proteins are very similar. Residues which have been shown to be important for the interaction of β'-COP with dilysine motifs [8] are conserved among the three β'-COP Arabidopsis paralogs, and thus one would not expect differential cargo binding among these paralogs. This may explain why β'1β'3-cop and β'2β'3-cop, although they showed different phenotypes under standard growth conditions, both showed mislocalization of p24δ5. The different phenotypes could be explained by different expression patterns of the β'-COP isoforms, as described above. However, it cannot be discarded that the different β'-COP isoforms have subsets of specific cargoes that could be responsible for the different observed phenotypes.
Altogether, our findings support an essential role of β'1-COP during seedling development. Future experiments should be performed to determine whether this role is due to its tissue or/and development pattern of expression or to a unique function of the β'1-COP isoform.

Data Availability Statement:
The data presented in the current study are available upon request to the corresponding authors.