A role for seed storage proteins in Arabidopsis seed longevity

Abstract

Proteomics approaches have been a useful tool for determining the biological roles and functions of individual proteins and identifying the molecular mechanisms that govern seed germination, vigour and viability in response to ageing. In this work the dry seed proteome of four Arabidopsis thaliana genotypes, that carry introgression fragments at the position of seed longevity quantitative trait loci and as a result display different levels of seed longevity, was investigated. Seeds at two physiological states, after-ripened seeds that had the full germination ability and aged (stored) seeds of which the germination ability was severely reduced, were compared. Aged dry seed proteomes were markedly different from the after-ripened and reflected the seed longevity level of the four genotypes, despite the fact that dry seeds are metabolically quiescent. Results confirmed the role of antioxidant systems, notably vitamin E, and indicated that protection and maintenance of the translation machinery and energy pathways are essential for seed longevity. Moreover, a new role for seed storage proteins (SSPs) was identified in dry seeds during ageing. Cruciferins (CRUs) are the most abundant SSPs in Arabidopsis and seeds of a triple mutant for three CRU isoforms (crua crub cruc) were more sensitive to artificial ageing and their seed proteins were highly oxidized compared with wild-type seeds. These results confirm that oxidation is involved in seed deterioration and that SSPs buffer the seed from oxidative stress, thus protecting important proteins required for seed germination and seedling formation.

Keywords: Arabidopsis; carbonylation; proteomics; reactive oxygen species; seed longevity; seed storage proteins..

Fig. 1.

Seed germination after seed dry storage. The germination (%) of Ler and the near-isogenic lines NILGAAS1, NILGAAS2 and NILGAAS5 were analysed for after-ripened seeds (open bars) and naturally-aged seeds after four years of storage (filled bars). Averages of four biological replicates with standard errors are presented. The asterisks indicate significant differences between aged NILs and Ler (P<0.05).

Fig. 2.

2D gel separation of seed proteins and the effect of ageing on seed protein abundance. (A) 2D gel of total soluble proteins from dry seeds stained with silver nitrate. The areas indicated on the gel (1 and 2) are enlarged in panels B and C. (B) Area 1 selected on the gel depicting the abundance of protein spot ID1345 that contains RPS12C and TPX1 proteins for the four genotypes (Ler and the near-isogenic lines NILGAAS1, NILGAAS2 and NILGAAS5) at two physiological states [after-ripened (AR) and aged]. (C) Area 2 showing the change in abundance of protein spot ID0667, corresponding to VTE1 protein, for the four genotypes at two physiological states. Arrows indicate the position of the proteins.

Fig. 3.

Principal component analysis (PCA) for proteome profiles of Ler and the near-isogenic lines NILGAAS1, NILGAAS2 and NILGAAS5. PCA was performed on the differentially accumulated protein spots in the seven comparisons (n=309).

Fig. 4.

Classification of differentially expressed protein spots. (A) Intersection of proteins that were differentially expressed between the two physiological states, aged versus after-ripened (AR), within Landsberg erecta (Ler) and the near-isogenic lines NILGAAS1, NILGAAS2 or NILGAAS5 (n=247). (B) Intersection of proteins that were differentially expressed between genotypes, each NIL versus Ler (n=74). (C, D, E) Intersection of proteins that were differential expressed in the genotypes NILGAAS1, NILGAAS2 and NILGAAS5 respectively in the earlier comparisons (as mentioned in A and B).

Fig. 5.

The effect of seed storage proteins (SSPs) on seed longevity and seed dormancy.(A) Seed longevity presented as germination (%) of different SSP knock-out lines was measured after 10 d of artificial ageing. The lines include the wild-type Col, as well as single (crua, aBC; crub, AbC and cruc, ABc), double (crub cruc, Abc; crua cruc, aBc and crua crub, abC) and triple (crua crub cruc, abc) knock-out lines of cruciferins, and an RNAi napin line that is depleted of napins. (B) Seed dormancy presented as days of seed dry storage required to reach 50% germination (DSDS50) of Col and different SSP knock-out lines.

Fig. 6.

Protein carbonylation of seed proteins in after-ripened (AR) artificially aged seeds (Aged). (A) 1D gel electrophoresis stained with Coomassie Brilliant Blue of total seed protein extracts from Col, the triple cruciferin mutant abc (crua crub cruc) and the napin mutant (RNAi-napin). (B) Carbonylated proteins as detected by immunodetection of protein-bound DNP after derivatization with hydrazine.