α2-COP is involved in early secretory traffic in Arabidopsis and is required for plant growth

Abstract

COP (coat protein) I-coated vesicles mediate intra-Golgi transport and retrograde transport from the Golgi to the endoplasmic reticulum. These vesicles form through the action of the small GTPase ADP-ribosylation factor 1 (ARF1) and the COPI heptameric protein complex (coatomer), which consists of seven subunits (α-, β-, β'-, γ-, δ-, ε- and ζ-COP). In contrast to mammals and yeast, several isoforms for coatomer subunits, with the exception of γ and δ, have been identified in Arabidopsis. To understand the role of COPI proteins in plant biology, we have identified and characterized a loss-of-function mutant of α2-COP, an Arabidopsis α-COP isoform. The α2-cop mutant displayed defects in plant growth, including small rosettes, stems and roots and mislocalization of p24δ5, a protein of the p24 family containing a C-terminal dilysine motif involved in COPI binding. The α2-cop mutant also exhibited abnormal morphology of the Golgi apparatus. Global expression analysis of the α2-cop mutant revealed altered expression of plant cell wall-associated genes. In addition, a strong upregulation of SEC31A, which encodes a subunit of the COPII coat, was observed in the α2-cop mutant; this also occurs in a mutant of a gene upstream of COPI assembly, GNL1, which encodes an ARF-guanine nucleotide exchange factor (GEF). These findings suggest that loss of α2-COP affects the expression of secretory pathway genes.

Keywords: Arabidopsis; COPI; COPII; Golgi apparatus; coat protein; p24 family protein; α1-COP; α2-COP; SEC31.

Fig. 1.

Characterization of the α1-cop-1 mutant. A. Diagram of the α1-COP gene and localization of the T-DNA insertion (triangle) in the α1-cop-1 mutant. Black boxes represent coding regions and grey boxes represent 5’ UTR and 3’ UTR regions. The positions of RPα1, LPα1, α125 and α123 primers are shown. B. sqRT-PCR analysis to show the absence of α1-COP mRNA in the α1-cop-1 mutant. Total RNA from 7-day-old seedlings of the mutant and wild type (Col-0) were used for the RT-PCR. For PCR, α1-COP gene specific primers, RPα1/LPα1, were used with 36 PCR cycles. ACT7 was used as a control with 22 cycles. C. α1-cop-1 mutant did not show a phenotype different from wild type.

Fig. 2.

Characterization of α2-cop mutants. A. Diagram of the α2-COP gene and localization of the T-DNA insertion (triangles) in the α2-cop mutants. Black boxes represent coding regions and grey boxes represent 5’ UTR and 3’ UTR regions.The positions of RPGα2, LPGα2, α125 and α123 primers are shown. B. sqRT-PCR analysis to show the absence of α2-COP mRNA in the α2-cop mutants. Total RNA from 7-day-old seedlings of the mutants and wild type (Col-0) were used for the RT-PCR. For PCRs, gene specific primers and 36 PCR cycles were used (Supplementary Table S1). ACT7 was used as a control with 22 PCR cycles. C. Phenotype of 4-week-old (left) and 7-day-old (right) seedlings of wild type and α2-cop-3 mutant. D. Rescue of the growth phenotype of α2-cop-3 by transformation with a HA tagged α2-COP cDNA construct. Phenotypes of 7-day-old seedlings (left) and 50-day-old plants (middle) of wild-type (Col-0), α2-cop-3 and α2-cop-3 complemented with α2-COP-HA. Western blot analysis, using a HA antibody, of the two independent lines of α2-cop-3 transformed with α2-COP-HA.

Fig. 3.

Expression levels of α-COP in α1-cop-1 and α2-cop-3 mutants. sqRT-PCR analysis of α-COP with α125 and α123 primers. Total RNA was isolated from 7-day-old seedlings of wild type (Col-0), α1-cop-1 and α2-cop-3 mutants. ACT7 was used as a control with 22 PCR cycles. A. Total α-COP expression in wild type and α1-cop-1 mutant. B. Total α-COP expression in wild type and α2-cop-3 mutant. C. Quantification of the experiments shown in A and B from three biological samples. Values were normalized against the α-COP fragment band intensity in wild type that was considered to be 100%. Error bars represent SEM. D. Western blot analysis of total protein extracts from 7-day-old seedlings of wild type, α1-cop-1 and α2-cop-3 mutants using an N-terminal α-COP peptide antibody to detect both isoforms. 10 μg of total protein was loaded in each lane. GAPC was used as a loading control.

Fig. 4.

α2-cop-3 mutant shows abnormal distribution of RFP- p24δ5. Confocal laser scanning microscopy of epidermal cells of 4.5-DAG cotyledons. RFP-p24δ5 mainly localized to the ER network in wild type plants (Col-0). In contrast, it was mostly found in punctate structures, which often appeared in clusters, in the α2-cop mutant. Scale bars, 10 µm.

Fig. 5.

α2-cop-3 mutant shows abnormal distribution of the Golgi marker ST-YFP. Confocal laser scanning microscopy of epidermal cells of cotyledons 4.5 days after germination. The Golgi marker ST-YFP partially localised to the ER network and to clusters of punctate structures in the a2-cop-3 mutant. Scale bar, 10 µm.

Fig. 6.

Alteration of Golgi morphology of cotyledon cells in the α2-cop-3 mutant. A. Chemically fixed cotyledon cells from 4-day-old wild type (Col-0) or α2-cop mutant seedlings. Scale bars, 200 nm. B. High-pressure frozen cotyledon cells from from 4-day-old α2-cop mutant seedlings. G, Golgi; V, vesicle; MVB, multivesicular body. Scale bars, 500 nm.

Fig. 7.

Expression of specific genes in α1-cop-1, α2-cop-3 and gnl1 mutants. A. sqRT-PCR validation of the microarray data performed for four genes whose expression changed in the α2-cop-3 mutant. Total RNA was extracted from 4-day-old seedlings. Specific primers were used and ACT7 was used as a control. B. RT-qPCR analysis of SEC31A and SEC31B expression in α1-cop-1 and α2-cop-3 mutants. Total RNA was extracted from 7-day-old seedlings. The mRNA was analyzed by RT-qPCR with specific primers and normalized to UBQ10 expression. Results are from two 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 ± SE of the two biological samples. C. sqRT-PCR analysis of COPII subunit genes in the α2-cop-3 mutant. Total RNA was extracted from 4-day-old seedlings. Specific primers were used and ACT7 was used as a control. D. sqRT-PCR analysis of SEC31A, BIP3 and PILS4, which showed altered expression in α2-cop-3, in gnl1 (SALK_091078C). Total RNA was extracted from 4-day-old seedlings. Specific primers were used and ACT7 was used as a control. The pattern of expression of all three genes is similar in both α2-cop-3 and gnl1. All specific primers are shown in Supplementary Table S1.