Nutrient deficiency stresses often occur simultaneously in soil.  Thus, it’s necessary to investigate the mechanisms underlying plant responses to multiple stresses through identification of some key stress-responsive genes.  Quantitative real-time PCR (qRT-PCR) is essential for detecting the expression of the interested genes, of which the selection of suitable reference genes is a crucial step before qRT-PCR.  To date, reliable reference genes to normalize qRT-PCR data under different nutrient deficiencies have not been reported in plants.  In this study, expression of ten candidate reference genes was detected in leaves and roots of rapeseed (Brassica napus L.) after implementing different nutrient deficiencies for 14 days.  These candidate genes, included two traditionally used reference genes and eight genes selected from an RNA-Seq dataset.  Two software packages (GeNorm, NormFinder) were employed to evaluate candidate gene stability.  Results showed that VHA-E1 was the highest-ranked gene in leaves of nutrient-deficient rapeseed, while VHA-G1 and UBC21 were most stable in nutrient-deficient roots.  When rapeseed leaves and roots were combined, UBC21, HTB1, VHA-G1 and ACT7 were most stable among all samples.  To evaluate the stabilities of the highest-ranked genes, the relative expression of two target genes, BnTrx1;1 and BnPht1;3 were further determined.  The results showed that the relative expression of BnTrx1;1 depended on reference gene selection, suggesting that it’s necessary to evaluate the stability of reference gene prior to qRT-PCR.  This study provides suitable reference genes for gene expression analysis of rapeseed responses to different nutrient deficiencies, which is essential for elucidation of mechanisms underlying rapeseed responses to multiple nutrient deficiency stresses.

In order to understand the response of nitrate metabolism in seedlings of oilseed rape (Brassica napus L.) to low oxygen stress (LOS), two cultivars were studied at different light, LOS time and exogenous nitrate concentrations under hydroponic stress. Results show that N-uptake and dry matter of rape seedlings were decreased after LOS stress while nitrate accumulation (NA) under LOS was induced by darkness. Nitrate accumulation peaked at 3 d while root activity (RA, defined as dehydrogenase activity) decreased with prolonged waterlogging exposure. Exogenous nitrate significantly elevated NA and RA. Tungstate (TS) and LOS inhibited nitrate reductase (NR) activity while NR transcription and activity were enhanced by exogenous nitrate. Low oxygen stress stimulated the activity of superoxide dismutase (SOD) and peroxidase (POD) slightly, but inhibited that of catalase (CAT). B. napus L. Zhongshuang 10 (ZS10), a LOS tolerant cultivar, displayed smaller decrease upon dry matter under LOS, higher NA in darkness and lower NA in light than B. napus L. Ganlan CC (GAC), a LOS sensitive variety. ZS10 had lower NA and higher RA after waterlogging and exogenous nitrate treatment, and higher NR activity under TS inhibition than GAC, but the activity of antioxidant enzymes did not change under LOS. The results indicate that nitrate metabolism involved tolerance of rape seedlings to LOS, with lower accumulation and higher reduction of nitrate being related to higher LOS tolerance of rape seedlings exposed to waterlogging.