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

Abstract

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.

Keywords: Arabidopsis; coat protein I (COPI); isoforms; plant growth; α-COP; β’-COP.

Figure 1

Expression patterns of β’1-COP, β’2-COP and β’3-COP. (A) Developmental stage-specific expression pattern in Arabidopsis thaliana. Seedlings, rosette leaves, floral organs and siliques are sequentially marked from left to right. “HIGH”, “MEDIUM” and “LOW” expressions were calculated by Afflymetrix Arabidopsis ATH1 genome array. The number of samples indicates RNA gene expression data collected by GENEVESTIGATOR (www.genevestigator.com, accessed on 23 November 2021). (B) Seed development expression pattern, from the globular embryo to the green cotyledons seed stage. β’1-COP shows the highest expression. Data collected and image generated by AtGenExpress eFP (http://bar.utoronto.ca/eplant, accessed on 15 December 2021) [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).

Figure 2

Relative expression levels of β’-COP genes in β’-cop mutants. RT–qPCR analysis was performed to characterize β’1-cop (A), β’2-cop (B) and β’3-cop (C) mutants. 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/wild type. Values represent mean ± s.e.m. of the three biological samples.

Figure 3

Characterization of β’1β’3-cop double mutants. (A) All the β’1β’3-cop homozygous double mutants show a dwarf phenotype at 7-day-old seedling stage. At later stages, the β’1β’3-cop-1 mutant also showed a dwarf phenotype with smaller rosette leaves, shorter stems and roots and reduced fertility (Supplementary Figure S3). No homozygous lines could be obtained of β’1β’3-cop-2 and β’1β’3-cop-3 mutants as they were only viable as seedlings and failed to develop beyond the seedling stage. White arrows point β’1β’3-cop-2 and β’1β’3-cop-3 homozygous seedlings obtained from seeds of β’1-cop-1/β’1-COP β’3-cop-2/β’3-cop-2 plants and β’1-cop-1/β’1-cop-1 β’3-COP/β’3-cop-3 plants, respectively. (B) RT–qPCR analysis show the expression levels of the three β’-COP genes in β’1β’3-cop double mutants relative to the wild type (Col-0). (C) β’1β’3-cop-1, β’1β’3-cop-2 and β’1β’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 wild type (Col-0) and homozygous mutants (β’1β’3-cop-2 and β’1β’3-cop-3 were selected by size). 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.

Figure 4

Characterization of β’2β’3-cop double mutants. (A) All the β’2β’3-cop double mutants show a wild type phenotype at 7-day-old seedling stage. (B) RT–qPCR analysis show the expression levels of the three β’-COP genes. (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.

Figure 5

Expression levels of coatomer subunit α-COPI in β’1β’3-cop-1 and β’2β’3-cop-2 mutants. (A) Western blot analysis of cytosol protein extracts from cotyledon of 7-day-old seedlings of wild type, β’1-cop-1, β’2-cop-1, β’3-cop-1 and β’3-cop-2 mutants using mammalian β’-COP and α-COP N-terminal peptide antibodies [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’3-cop-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’3-cop-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.

Figure 6

β’-cop double mutants show abnormal distribution of RFP–p24δ5, a COPI dilysine cargo. 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.

Figure 7

Trafficking of p24δ5 in wild-type and β’-COP double mutant plants. (A) In wild-type plants, p24δ5 mainly localizes in the ER at steady-state due to efficient, COPI-dependent, Golgi-to-ER transport. (B) In β’-COP double mutants, p24δ5 is not efficiently retrieved from the Golgi apparatus and thus follows a default pathway to the vacuole, where the luminal part of the protein (including RFP in the case of RFP–p24δ5) is cleaved and released to the vacuole lumen.