Quantitative RT–PCR Platform to Measure Transcript Levels of C and N Metabolism-Related Genes in Durum Wheat: Transcript Profiles in Elevated [CO2] and High Temperature at Different Levels of N Supply

47 páginas, 4 tablas, 2 figuras. -- This is a pre-copyedited, author-produced PDF of an article accepted for publication in Plant and Cell Physiology following peer review. The version of record [Rubén Vicente, Pilar Pérez, Rafael Martínez-Carrasco, Björn Usadel, Svetla Kostadinova, and Rosa Morcuende. Quantitative RT–PCR Platform to Measure Transcript Levels of C and N Metabolism-Related Genes in Durum Wheat: Transcript Profiles in Elevated [CO2] and High Temperature at Different Levels of N Supply Plant Cell Physiol (2015) 56 (8): 1556-1573, 2015 doi:10.1093/pcp/pcv079 ] is available online at: http://pcp.oxfordjournals.org/content/56/8/1556

Only limited public transcriptomics resources are available for durum wheat and its responses to environmental changes. We developed a quantitative reverse transcription–PCR (qRT–PCR) platform for analysing the expression of primary C and N metabolism genes in durum wheat in leaves (125 genes) and roots (38 genes), based on available bread wheat genes and the identification of orthologs of known genes in other species. We also assessed the expression stability of seven reference genes for qRT–PCR under varying environments. We therefore present a functional qRT–PCR platform for gene expression analysis in durum wheat, and suggest using the ADP-ribosylation factor as a reference gene for qRT–PCR normalization. We investigated the effects of elevated [CO2] and temperature at two levels of N supply on C and N metabolism by combining gene expression analysis, using our qRT–PCR platform, with biochemical and physiological parameters in durum wheat grown in field chambers. Elevated CO2 down-regulated the photosynthetic capacity and led to the loss of N compounds, including Rubisco; this effect was exacerbated at low N. Mechanistically, the reduction in photosynthesis and N levels could be associated with a decreased transcription of the genes involved in photosynthesis and N assimilation. High temperatures increased stomatal conductance, and thus did not inhibit photosynthesis, even though Rubisco protein and activity, soluble protein, leaf N, and gene expression for C fixation and N assimilation were down-regulated. Under a future scenario of climate change, the extent to which C fixation capacity and N assimilation are down-regulated will depend upon the N supply

This work was supported by the Spanish National R&D&i Plan of the Ministry of Economy and Competitiveness (grant numbers AGL2006-13541-C02-02, AGL2009-11987, BES-2010-031029 to R.V.)

Peer reviewed