Misregulation of ER-Golgi Vesicle Transport Induces ER Stress and Affects Seed Vigor and Stress Response

Abstract

Seeds of higher plants accumulate numerous storage proteins to use as nitrogen resources for early plant development. Seed storage proteins (SSPs) are synthesized as large precursors on the rough endoplasmic reticulum (rER), and are delivered to protein storage vacuoles (PSVs) via vesicle transport, where they are processed to mature forms. We previously identified an Arabidopsis ER-localized tethering complex, MAG2 complex, which might be involved in Golgi to ER retrograde transport. The MAG2 complex is composed of 4 subunits, MAG2, MIP1, MIP2, and MIP3. Mutants with defective alleles for these subunits accumulated SSP precursors inside the ER lumen. Here, we report that the mag2-1 mip3-1 and mip2-1 mip3-1 double mutant have more serious vesicle transport defects than the mag2-1, mip2-1, and mip3-1 single mutants, since they accumulate more SSP precursors than the corresponding single mutants, and ER stress is more severe than the single mutants. The mag2-1 mip3-1 and mip2-1 mip3-1 double mutants show growth and developmental defects rather than the single mutants. Both single and double mutant seeds are found to have lower protein content and decreased germinating vigor than wild type seeds. All the mutants are sensitive to abscisic acid (ABA) and salt stress, and exhibit alteration in ABA signaling pathway. Our study clarified that ER-Golgi vesicle transport affects seed vigor through controlling seed protein quality and content, as well as plant response to environmental stress via influencing ABA signaling pathway.

Keywords: ABA signaling; ER-Golgi vesicle transport; MAG2 complex; plant stress response; seed vigor.

FIGURE 1

The mag2-1 mip3-1 double mutant exhibits dwarf phenotype. (A) Immunoblot of maturing seeds from the wild type (Col-0, the same below) and mag2-1 mip3-1 double mutant with anti-MAG2 antibody (left panel). Right panel indicates expression level of MIP3 in 9-day-old seedlings of wild type and mag2-1 mip3-1 double mutant. RT-PCR was performed using MIP3 specific primers (Li et al., 2013). (B) Immunoblot of 7-day-old seedlings from the wild type and mip2-1 mip3-1 double mutant with anti-MIP2 antibody (left panel). Right panel indicates expression level of MIP3 in 7-day-old seedlings of wild type and mip2-1 mip3-1 double mutant. (C) 7-day-old seedlings of wild type, mag2-1 mip3-1 and mip2-1 mip3-1 double mutants (left panel) and root length statistics (right panel). n = 30, two independent experiments per sample. (D) 41-day-old plants of wild type and mag2-1 mip3-1. (E) 21-day-old plants of wild type and mip2-1 mip3-1.

FIGURE 2

Defects in storage protein trafficking for the mag2-1, mip3-1 and mag2-1 mip3-1 mutants. (A,B) Immunoblot of dry seeds of wild-type and the mutants with anti-12S and anti-p2S antibodies. Arrows indicate SSP precursors. p12S, pro12S globulin; p2S, pro2S albumin; 12S, 12S globulin; 2S, 2S albumin. (C) Seed protein profile. Coomassie brilliant blue (CBB) staining of an SDS–PAGE gel for seed proteins. Abnormally accumulated SSP precursors are indicated by arrows. (D) Seed grains of wild type and the mutants. (E) Thousand-grain-weight (TGW) for wild type and the mutant seeds. (F) Protein content of one thousand grains is measured by BCA Protein Quantification Kit (ml410021, Mlbio, China).

FIGURE 3

ER stress is induced in the mutants. (A) Immunoblot of dry seeds of wild type, mag2-1, mip3-1 and mag2-1 mip3-1, with anti-BiP1/2 antibodies. Statistics for BiP1/2 protein accumulation in seeds are shown in numbers below the bands BiP/control (ACT or TUB, actin or tubulin) band concentration, measured by ImageJ, the same below). Three independent experiments per sample. (B) BiP3 expression was detected by RT-PCR on 9-day-old seedlings using BiP3 specific primers. Statistics for BIP3 expression level are shown in numbers below the bands. n = 3. Three independent experiments per sample. (C) Quantitative analysis of germination ratio (Green cotyledon ratio, the same below) of wild type, mag2-1, mip3-1 and mag2-1 mip3-1 on 1/2MS medium (left) or 1/2MS medium with 1.2 mM DTT (right). n = 72, three independent experiments per sample. (D) Root length change fold (treated/non-treated) of 7-old-seedlings of wild type, mag2-1 mip3-1 and mip2-1 mip3-1 on 1/2MS medium (non-treated) and 1/2MS medium with 0.15 mM DTT (treated). (E) Immunoblot on DTT treated 7-day-old seedlings of wild type, mag2-1, mip3-1 and mag2-1 mip3-1 on 1/2MS medium (left panel) or 1/2MS medium with 1.2 mM DTT (right panel) with anti-BiP1/2 antibody. Statistics of BiP1/2 protein level are shown below the bands. Change fold (treated/non-treated, the same below) is indicated in the lowest panel.

FIGURE 4

Vigor of aging mutant seeds decreased rapidly. Germination ratio statistics for seeds from different storage time on the 7th day after germination (DAG). n = 50, three independent experiments per sample. The seeds used in this experiment were collected from 2013 to 2017.

FIGURE 5

Mutant seeds have lower protein carbonylation. (A) Quantification of seed protein carbonylation of wild type and mutants. (B) Carbonylated proteins were detected by immunodetection of protein-bound DNP after derivatization with hydrazine with anti-Dinitrophenol antibody (left panel). Right panel is CBB staining of a SDS–PAGE gel for total protein extracts from wild type and mutant seeds. Statistics of the bands are shown in numbers below the bands.

FIGURE 6

ABA and NaCl treatments result in a greater germination ratio decline in mutants than in wild type. Quantitative analysis of germination ratio of wild type and the mutants on 1/2MS medium (A,D), 1/2MS medium with 0.3 μM ABA (B,E) or with 130 mM NaCl (C,F). n = 50, three independent experiments per sample.

FIGURE 7

ABI3 and ABI4 expression level altered in mutants. ABI3 and ABI4 expression level in wild type and mag2-1 mip3-1 under ABA treatment (A) or under NaCl treatment (B) were determined by qRT-PCR on 10-day-old seedlings. For qRT-PCR, three repeats per experiment, three experiments per sample. (C) Immunoblot on the ABA and NaCl treated 10-day-old seedlings with anti-ABI5 antibody. Statistics of ABI5 protein level are shown below the bands.

FIGURE 8

ABI5 protein accumulation level altered in mutant seeds. Immunoblot with anti-ABI5 antibody on maturing seeds from the No. 2 and No. 3 siliques below the inflorescences (A), dry seeds (B) and imbibing seeds after 48 h imbibition (C). Lower panels are statistics of upper panels.