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.

The investigation was carried out to study the response of winter rapeseed to potassium (K) feritlization and the critical soil available K level for current winter rapeseed production in the Yangtze River Valley (YRV) of China. A total of 132 field experiments were conducted in fields of farmers in the major winter rapeseed growing areas in YRV in 2000/2001 and 2004/2005 to 2006/2007 during growing season. Results of these field experiments showed that the average rapeseed yield increment resulting from 100 kg K ha^-1 application was 358 kg ha^-1, an increase over the control CK (no K) of 18.0% in 2005/2006 and 2006/2007. The average internal use efficiency (IE) of K was higher in the CK treatment (21.9 kg grain, kg-1 K uptake) than in the +K (100 kg K ha^-1) treatment (17.7 kg grain, kg^-1 K uptake). Winter rapeseed required 68.1 kg of K to produce 1 000 kg seed. The recovery efficiency of K fertilizer in rapeseed production averaged 39.3%. The K balance was negative, with an average net removal of 117.6 kg K ha^-1 in the CK treatment annually, and 56.8 kg K ha^-1 in the +K treatment. The results indicated that there was a significant negative relationship between yield increments by K application and soil available K content. Based on the relative yield of CK/+K at 90% level, the critical level of soil available K(NH₄OA_c-extractable K) was 135 mg kg^-1.