Transcript and protein profiling identify candidate gene sets of potential adaptive significance in New Zealand Pachycladon

Abstract

Background: Transcript profiling of closely related species provides a means for identifying genes potentially important in species diversification. However, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear. In the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three Pachycladon species. We compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species.

Results: Evidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis (myrosinase-associated proteins) and biosynthesis (methylthioalkylmalate isomerase and dehydrogenase), the interconversion of carbon dioxide and bicarbonate (carbonic anhydrases), water use efficiency (ascorbate peroxidase, 2 cys peroxiredoxin, 20 kDa chloroplastic chaperonin, mitochondrial succinyl CoA ligase) and others (glutathione-S-transferase, serine racemase, vegetative storage proteins, genes related to translation and photosynthesis). Differences in glucosinolate hydrolysis products were directly confirmed. Overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. Protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. The similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not.

Conclusions: We have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three Pachycladon species. This approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study. Our results indicate that protein profiling provides a valuable tool for validating transcript profiles in studies of adaptive divergence.

Figure 1

Overlap in differential expression patterns from both transcript and protein profiling. Numbers of differentially expressed genes in P. cheesemanii (CH), P. exile (EX) and P. novae-zelandiae (NZ) identified by transcript (T) profiling, protein profiling (P) or both (TP). Differential expression is defined as significantly higher or lower expression in one species compared with the other two combined (panel A - 6 group comparisons) or separately (panel B - 6 pair wise comparisons) and was determined by linear model analysis (transcripts) or Wilcoxon rank and t-tests (proteins). Numbers are corrected for relative sizes of transcript and protein data sets. In other words, only a subset of all differentially expressed transcripts and proteins is given. This subset consists of genes found amongst the 1074 loci surveyed by both transcript and protein profiling. For a summary of all differentially expressed transcripts and proteins refer to table 2. Upper percentages depict the proportion of transcripts confirmed by protein analysis and lower percentages depict the proportion of proteins confirmed by transcript analysis (see text for details). Statistically significant overlap (p value < 0.05%) is denoted with a star. For locus IDs, gene descriptions, transcript and protein expression statistics of genes identified as being up-regulated by both methods (TP) in CH, EX and NZ see table 3.

Figure 2

Hierarchical clustering of glucosinolate profiles. Fourteen compounds were identified across individuals of P. cheesemanii (CH, n = 12), P. exile (EX, n = 13) and P. novae-zelandiae (NZ, n = 12). Data used for hierarchical clustering represent proportions from total contents; for comparisons of concentrations see additional file 2, figure S1. Note that CH and NZ share their two major compounds allyl and S-2OH3-butenyl glucosinolate despite being less closely related than CH and EX. *The glucosinolate profiles for P. novae-zelandiae and P. cheesemanii were independently confirmed during the analysis of glucosinolate breakdown products in both species (table 5). Abbreviations: 3MTP, 3-methylthiopropyl glucosinolate; 4MTB, 4-methylthiobutyl glucosinolate; 3MSOP, 3-methylsulfinylpropyl glucosinolate; 4MSOB, 4-methylsulfinylbutyl glucosinolate; allyl, 2-propenyl glucosinolate; 3-butenyl, 3-butenyl glucosinolate; S-2OH3-butenyl, S-2-hydroxy-3-butenyl glucosinolate; 6MSOH, 6-methylsulfinylhexyl glucosinolate; 7MTH, 7-methylthioheptyl glucosinolate 7MSOH, 7-methylsulfinylheptyl glucosinolate; 8MSOO, 8-methylsulfinyloctyl glucosinolate; 1MOI3M, 1-methoxy-indolyl-3-methyl glucosinolate; 4OHI3M, 4-hydroxy-indolyl-3-methyl glucosinolate, 4MOI3M, 4-methoxy-indolyl-3-methyl glucosinolate.