Protein nonadditive expression and solubility contribute to heterosis in Arabidopsis hybrids and allotetraploids

Hybrid vigor or heterosis has been widely applied in agriculture and extensively studied using genetic and gene expression approaches. However, the biochemical mechanism underlying heterosis remains elusive. One theory suggests that a decrease in protein aggregation may occur in hybrids due to the presence of protein variants between parental alleles, but it has not been experimentally tested. Here, we report comparative analysis of soluble and insoluble proteomes in Arabidopsis intraspecific and interspecific hybrids or allotetraploids formed between A. thaliana and A. arenosa. Both allotetraploids and intraspecific hybrids displayed nonadditive expression (unequal to the sum of the two parents) of the proteins, most of which were involved in biotic and abiotic stress responses. In the allotetraploids, homoeolog-expression bias was not observed among all proteins examined but accounted for 17-20% of the nonadditively expressed proteins, consistent with the transcriptome results. Among expression-biased homoeologs, there were more A. thaliana-biased than A. arenosa-biased homoeologs. Analysis of the insoluble and soluble proteomes revealed more soluble proteins in the hybrids than their parents but not in the allotetraploids. Most proteins in ribosomal biosynthesis and in the thylakoid lumen, membrane, and stroma were in the soluble fractions, indicating a role of protein stability in photosynthetic activities for promoting growth. Thus, nonadditive expression of stress-responsive proteins and increased solubility of photosynthetic proteins may contribute to heterosis in Arabidopsis hybrids and allotetraploids and possibly hybrid crops.

Supplementary Fig. 2. Reproducibility of protein abundances in intraspecific hybrids.
Pearson correlation of protein abundances for all intraspecific hybrid samples.There is a high degree of reproducibility between biological replicates, with the insoluble fractions showing lower levels of correlation with soluble and whole cell extract fractions.

Supplementary Fig. 1 .
Log 2 (normalized precursor abundance) values of the peptides among all samples analyzed.Precursor ion abundance is calculated based on the chromatogram peak area of the precursor ion.(A) Log 2 (normalized precursor abundance) of all peptides identified in the intraspecific hybrid samples (B) Log 2 (normalized precursor abundance) of all peptides identified in the interspecific polyploid samples.
x C o l I n s o l u b l e C 2 4 x C o l S o l u b l e C 2 4 x C o l W C E C o l I n s o l u b l e C o l S o l u b l e C o l W C E C o l x C 2 4 I n s o l u b l e C o l x C 2 4 S o l u b l e C o l x C 2 Reproducibility of protein abundance in allotetraploids.Pearson correlation of protein abundances for all allotetraploid samples.There is a high degree of reproducibility between biological replicates, with the insoluble fractions showing lower levels of correlation with soluble and whole cell extract fractions.
Overlap of the proteins that were differentially expressed between the parents with the non-additively expressed proteins.The proteins differentially expressed between the progenitors of each hybrid or allotetraploid were overlapped with the non-additively expressed proteins in (A) All738, (B) A. suecica, (C) F 1 (Col x C24), and (D) F 1 (C24 x Col).Enrichment of soluble fractions in cytosolic proteins and of insoluble fractions in proteins localized to membranes.GO cellular component enrichment of proteins significantly enriched in the insoluble and soluble fractions in both experiments.The soluble fraction shows strong enrichment across several cellular components including the cytosol, whereas the insoluble fraction only shows enrichment for chloroplast proteins, particularly those in the thylakoid membrane.Supplementary Fig.6.Evaluation of homoeolog-specific expression found no obvious homoeolog-specific expression of more soluble proteins in Arabidopsis allotetraploids.The distribution of TANGO scores of the A. thaliana and A. arenosa homoeologs of all proteins that display homoeolog biased expression in (A) Allo738 and (B) A. suecica and the distribution of instability scores of the A. thaliana and A. arenosa homoeologs of all proteins that display homoeolog biased expression in (C) Allo738 and (D) A. suecica.For each graph, grey boxes divide graph into proteins that displayed either A. thaliana or A. arenosa subgenome bias, and the two distributions within these panels indicate the distribution of TANGO or instability scores for the A. arenosa and A. thaliana homoeologs of the proteins that display this biased expression.
Evaluation of homoeolog-specific expression found no obvious homoeolog-specific expression of more soluble proteins in Arabidopsis allotetraploids.The distribution of TANGO scores of the A. thaliana and A. arenosa homoeologs of all proteins that display homoeolog biased expression in (A) Allo738 and (B) A. suecica and the distribution of instability scores of the A. thaliana and A. arenosa homoeologs of all proteins that display homoeolog biased expression in (C) Allo738 and (D) A. suecica.For each graph, grey boxes divide graph into proteins that displayed either A. thaliana or A. arenosa subgenome bias, and the two distributions within these panels indicate the distribution of TANGO or instability scores for the A. arenosa and A. thaliana homoeologs of the proteins that display this biased expression.