Ragweed (Ambrosia artemisiifolia) pollen allergenicity: SuperSAGE transcriptomic analysis upon elevated CO2 and drought stress

Abstract

Background: Pollen of common ragweed (Ambrosia artemisiifolia) is a main cause of allergic diseases in Northern America. The weed has recently become spreading as a neophyte in Europe, while climate change may also affect the growth of the plant and additionally may also influence pollen allergenicity. To gain better insight in the molecular mechanisms in the development of ragweed pollen and its allergenic proteins under global change scenarios, we generated SuperSAGE libraries to identify differentially expressed transcripts.

Results: Ragweed plants were grown in a greenhouse under 380 ppm CO2 and under elevated level of CO2 (700 ppm). In addition, drought experiments under both CO2 concentrations were performed. The pollen viability was not altered under elevated CO2, whereas drought stress decreased its viability. Increased levels of individual flavonoid metabolites were found under elevated CO2 and/or drought. Total RNA was isolated from ragweed pollen, exposed to the four mentioned scenarios and four SuperSAGE libraries were constructed. The library dataset included 236,942 unique sequences, showing overlapping as well as clear differently expressed sequence tags (ESTs). The analysis targeted ESTs known in Ambrosia, as well as in pollen of other plants. Among the identified ESTs, those encoding allergenic ragweed proteins (Amb a) increased under elevated CO2 and drought stress. In addition, ESTs encoding allergenic proteins in other plants were also identified.

Conclusions: The analysis of changes in the transcriptome of ragweed pollen upon CO2 and drought stress using SuperSAGE indicates that under global change scenarios the pollen transcriptome was altered, and impacts the allergenic potential of ragweed pollen.

Figure 1

Amount of PBS-soluble (a) and methanolic-extractable (b) phenolic metabolites in ragweed pollen. The separation was performed by RP-HPLC. The bars (N = 5) indicate SD and significant differences are indicated by an asterisk.

Figure 2

Venn diagram. Number of common and unique SuperSAGE sequence tags. For each sequence, the tag amount in the individual samples was analyzed. Sequences with ≥ 1 appearances in two, three or all of the samples are shown by individual overlapping regions. The total number of sequence tags per library is indicated. a reflects the distribution of sequence tags in the original dataset. b gives the distribution of sequenced tags filtered for tpm > 0.8. c indicates the sequence tag distribution for a stringently filtered dataset with the following criteria: tpm > 0.8; score of BLAST hit > 40; and removal of sequence tags without BLAST result (“no hit”).

Figure 3

Distribution of low- to very high-abundant sequence tags. The tags were found uniquely under control conditions (380 ppm CO₂), under elevated CO₂ (700 ppm CO₂), under CO₂ plus drought or under drought, or found to be common in all four libraries at one time. The data were analyzed for the different filter criteria indicated in the graph.

Figure 4

Quantitative real-time RT-PCR of selected ragweed allergens. The relative expression is indicated as fold change. The gene-specific primers are given in Additional file 8. As a reference gene, α-tubulin was used. The bars indicate SE and an asterisk indicates significant changes; N = 4 individual plants and three technical replicates.