Genome-wide characterization of LEA gene family reveals a positive role of BnaA.LEA6.a in freezing tolerance in rapeseed (Brassica napus L.)

Abstract

Background: Freezing stress is one of the major abiotic stresses that causes extensive damage to plants. LEA (Late embryogenesis abundant) proteins play a crucial role in plant growth, development, and abiotic stress. However, there is limited research on the function of LEA genes in low-temperature stress in Brassica napus (rapeseed).

Results: Total 306 potential LEA genes were identified in B. rapa (79), B. oleracea (79) and B. napus (148) and divided into eight subgroups. LEA genes of the same subgroup had similar gene structures and predicted subcellular locations. Cis-regulatory elements analysis showed that the promoters of BnaLEA genes rich in cis-regulatory elements related to various abiotic stresses. Additionally, RNA-seq and real-time PCR results indicated that the majority of BnaLEA family members were highly expressed in senescent tissues of rapeseed, especially during late stages of seed maturation, and most BnaLEA genes can be induced by salt and osmotic stress. Interestingly, the BnaA.LEA6.a and BnaC.LEA6.a genes were highly expressed across different vegetative and reproductive organs during different development stages, and showed strong responses to salt, osmotic, and cold stress, particularly freezing stress. Further analysis showed that overexpression of BnaA.LEA6.a increased the freezing tolerance in rapeseed, as evidenced by lower relative electrical leakage and higher survival rates compared to the wild-type (WT) under freezing treatment.

Conclusion: This study is of great significance for understanding the functions of BnaLEA genes in freezing tolerance in rapeseed and offers an ideal candidate gene (BnaA.LEA6.a) for molecular breeding of freezing-tolerant rapeseed cultivars.

Keywords: Brassica napus; Abiotic stress; Freezing stress; LEA (Late embryogenesis abundant).

Fig. 1

Phylogenetic tree of 357 LEA proteins from B. rapa (79), B. oleracea (79), B. napus (148) and Arabidopsis (51). A Maximum Likelihood phylogenetic tree was generated with full-length LEA protein sequences (1000 bootstrap replicates). The eight resulting groups (LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, SMP and DHN) are labeled by different colors

Fig. 2

Expression of BnaLEA genes in different tissues and organs of ZS11 (semi-winter type cv. Zhongshuang 11). Colored rectangles indicate expression levels of BnaLEA genes. Red means high expression, Blue means low expression. Color from red to blue represents descending log₂(TPM + 1)

Fig. 3

Expression of BnaLEA genes under different abiotic stresses treatments in leaves. Colored rectangles indicate expression levels of BnaLEA genes. Red means high expression, Blue means low expression. Color from red to blue represents descending log₂(TPM + 1)

Fig. 4

Expression patterns of BnaLEA genes under different plant hormones treatments in leaves. Colored rectangles indicate expression levels of BnaLEA genes. Red means high expression, Blue means low expression. Color from red to blue represents descending log₂(TPM + 1)

Fig. 5

qRT-PCR analysis of BnaA.LEA6.a gene in different tissues/organs and under cold and freezing stress. a The expression levels of the BnaA.LEA6.a gene in the root, stem, leaf, and seeds (at 20, 30, and 40 days after flowering) in the Brassica napus variety Zhongshuang 6. b The expression levels of BnaA.LEA6.a gene under cold (4℃) and freezing (-4℃) stress in B. napus. In group A, after 14 days of cold acclimation, plants were subjected to 12 h of low temperature at 4℃ and freezing stress at -4℃. In group B, plants were not cold-acclimated and were directly subjected to 12 h of treatment at 4℃ and freezing stress at -4℃. BnaActin (BnaA09G0588200ZS) was used as the endogenous reference gene. The relative transcript levels were averaged over the three technical replicates

Fig. 6

Phenotypes of the BnaA.LEA6.a-overexpression transgenic rapeseed lines and WT before and after freezing stress treatment. a 30-day-old WT and OE lines recovered to 4 d phenotype after 2 h treatment at -3.5 ℃. b The relative expression level of BnaA.LEA6.a-overexpression transgenic plants and WT. c The relative electrical conductivity (%) after freezing treatment. d Statistics on the survival rate of 30-day-old WT and OE lines treated at -3.5 ℃ for 2 h and recovered for 7 d. Data were expressed as the mean of triplicate values, and the error bar represented the SEM. Asterisks indicate statistically significant differences between transgenic lines and the corresponding WT plants at P ≤ 0.01(**), P ≤ 0.05(*)