Combining Physio-Biochemical Characterization and Transcriptome Analysis Reveal the Responses to Varying Degrees of Drought Stress in Brassica napus L

Abstract

Brassica napus L. has become one of the most important oil-bearing crops, and drought stress severely influences its yield and quality. By combining physio-biochemical characterization and transcriptome analysis, we studied the response of B. napus plants to different degrees of drought stress. Some physio-biochemical traits, such as fresh weight (FW), dry weight (DW), abscisic acid (ABA) content, net photosynthetic rate (Pn), stomatal conductance (g_s), and transpiration rate (Tr), were measured, and the total content of the epidermal wax/cutin, as well as their compositions, was determined. The results suggest that both stomatal transpiration and cuticular transpiration are affected when B. napus plants are subjected to varying degrees of drought stress. A total of 795 up-regulated genes and 1050 down-regulated genes were identified under severe drought stress by transcriptome analysis. Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) revealed that the up-regulated genes were mainly enriched in the stress response processes, such as response to water deprivation and abscisic acid, while the down-regulated genes were mainly enriched in the chloroplast-related parts affecting photosynthesis. Moreover, overexpression of BnaA01.CIPK6, an up-regulated DEG, was found to confer drought tolerance in B. napus. Our study lays a foundation for a better understanding of the molecular mechanisms underlying drought tolerance in B. napus.

Keywords: BnaCIPK6; Brassica napus; drought tolerance; physio-biochemical characterization; transcriptome.

Figure 1

Characterization of physio-biochemical traits responding to varying degrees of drought stress. (A) The response of B. napus to varying degrees of drought stress. Bar = 10 cm. (B) Aboveground fresh weight under varying degrees of drought stress. (C) Aboveground dry weight under varying degrees of drought stress. The data are the means ± standard deviation (n = 4). (D–G) Abscisic acid content (D), stomatal conductance (E), net photosynthetic rate (F), transpiration rate (G) under varying degrees of drought stress. LD, mild drought stress; MD, moderate drought stress; SD, severe drought stress; CK-LD, CK-MD, and CK-SD, the respective controls of LD, MD and SD. The data are the means ± standard deviation (n = 4). Different lowercase letters indicate significant differences, while the same letters indicate no significant difference (one–way ANOVA for multiple comparisons, p < 0.05).

Figure 2

Morphology observation and determination of epidermal wax content and composition under varying degrees of drought stress. (A–H) Scanning electron microscope observation of epidermal wax morphology under varying degrees of drought stress at the magnifications of 1000× g (A–D) and 4500× g (E–H). (I) Determination of total wax content under varying degrees of drought stress. (J) Quantitative analysis of wax components by GC-MS. The data are the means ± SD (n = 4). LD, mild drought stress; MD, moderate drought stress; SD, severe drought stress; CK-LD, CK-MD, and CK-SD, the respective controls of LD, MD, and SD. Statistical significance was determined by Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Quantitative analysis of cutin components by GC-MS under varying degrees of drought stress conditions. (A) Determination of total cutin content under varying degrees of drought stress. (B–G) Determination of primary alcohol content (B), unsaturated fatty acid content (C), saturated fatty acid content (D), hydroxy fatty acid content (E), dicarboxylic acids content (F), and unknown compounds content (G) under varying degrees of drought stress. LD, mild drought stress; MD, moderate drought stress; SD, severe drought stress; CK-LD, CK-MD and CK-SD, the respective controls of LD, MD, and SD. Statistical significance was determined by Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Transcriptome analysis of B. napus plants subjected to severe drought stress. (A) Gene ontology (GO) analysis of up-regulated differentially expressed genes (DEGs) under severe drought stress. (B) GO analysis of down-regulated DEGs under severe drought stress. (C) DEGs) involved in ABA biosynthesis and signal transduction pathways. (D) DEGs involved in the epidermal wax and cutin biosynthesis pathway. CK, control; SD, severe drought.

Figure 5

Over-expression of BnaA01.CIPK6 confers drought tolerance in B. napus. (A) Expression pattern analysis of BnCIPK6. (B) Relative expression level of BnaA01.CIPK6 gene in two BnaA01.CIPK6-overexpression lines, OE-7 and OE-9. (C) Western blot analysis of BnaA01.CIPK6 gene expression in BnaA01.CIPK6-overexpression (OE-7 and OE-9). (D) The response of BnaA01.CIPK6-overexpression plants (OE-7 and OE-9) to drought stress. Bar = 10 cm. (E) Survival rate of BnaA01.CIPK6-overexpression lines (OE-7 and OE-9) under drought stress. (F) Measurement of photosynthesis-related parameters of BnaA01.CIPK6-overexpression lines (OE-7 and OE-9) under drought stress. (G) Aboveground biomass of BnaA01.CIPK6-overexpression lines (OE-7 and OE-9) under drought stress. The data are the means ± standard deviation (n = 4). Statistical significance was assayed by Student’s t-test. * p < 0.05, ** p < 0.01.