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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (04): 483-492.doi: 10.3724/SP.J.1006.2018.00483


Enhanced Accumulation of BnA7HSP70 Molecular Chaperone Binding Protein Improves Tolerance to Drought Stress in Transgenic Brassica napus

Li-Li WAN1,*(), Zhuan-Rong WANG2, Qiang XIN2, Fa-Ming DONG2, Deng-Feng HONG2, Guang-Sheng YANG2   

  1. 1 Institute of Crop, Wuhan Academy of Agricultural Sciences, Wuhan 430065, Hebei, China
    2 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430065, Hebei, China
  • Received:2017-09-04 Accepted:2018-01-08 Online:2018-01-31 Published:2018-01-31
  • Contact: Li-Li WAN E-mail:wanlili13226@163.com
  • Supported by:
    This study was supported by Germplasm Innovation Foundation of Wuhan Academy of Agricultural Sciences (CX201710), the National Natural Science Foundation of China (31401413), and the High Technology Innovation Foundation of Hubei Province (2016ABA084).


The molecular chaperone binding protein gene participates in the constitutive function of plant growth and protects plant cells against stresses. In this study, we found that BnA7HSP70 overexpressed transgenic lines did not wilt and showed only a small decrease in water potential. However, the wild type lines showed a large decrease in leaf water potential. The transgenic plants had higher relative water content, better osmotic adjustment and less lipid membrane peroxidation. In addition, the leaves from the elevated levels of BnA7HSP70 in transgenic lines conferred tolerance to the glycosylation inhibitor tunicamycin during germination. BnA7HSP70 overexpression-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell and the reduced induction of the senescence-associated marker gene BnCNX1. These phenotypes were accompanied by a delay in the induction of the cell death marker genes BnNRP, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP (N-rich protein)-mediated signaling pathway. Enhanced expression of BnA7HSP70 delayed unfold protein response and NRP pathway mediated chlorosis and the appearance of senescence-associated markers BnLSC222 and BnLSC54 in Brassica napus. These results suggest that overexpression of BnA7HSP70 in Brassica napus alleviate ER stress and osmotic stress-integrating cell death response confronted with water stress.

Key words: BnA7HSP70, drought tolerance, Brassica napus, leaf senescence, antioxidant enzymes

Fig. 1

Subcellular localization of BnA7HSP70 and GFP fusion protein in Arabidopsis protoplasts. (A) Subcellular localization of full-length GFP fused with green fluorescent protein (GFP), the cytoplasm showed a green fluorescent signal at 488 nm. (B) The mesophyll cells showed a red fluorescent signal at 580 nm. (C) A bright-field image of protoplast cell; (D) A bright-field image and the merged image are shown at the bottom. Scale bars, 10 µm."

Fig. 2

BnA7HSP70 overexpressed (OE) plants confer tolerance under a restricted water regime and 20% PEG treatment(A) For the fast soil drying treatment, wild type (WT) and BnA7HSP70 overexpressed (OE) lines were allowed to reach four to five weeks leaves stage of development when drought was rapidly induced by withholding irrigation for 10 days. The stress condition was prolonged until the leaves of wild type plants completely wilted. (B) For the fast soil drying treatment, wild type (WT) and BnA7HSP70 overexpressed (OE) lines were allowed to reach four to five weeks leaves stage of development when drought was rapidly induced by withholding irrigation for 15 days. The stress condition was prolonged until the leaves of wild type plants completely wilted. (C) After rewatering for 3 days, most wild type plants were unable to recover, while OE plants survived continued to grow. (D) 40-day-old WT and OE transgenic plants grown in nutrient solution. (E) For drought stress, 40-day-old WT and OE transgenic plants were transferred into nutrient solution containing 20% (w/v) PEG-6000 for 48 h. The concentration of PEG was maintained daily by changing the nutrient solution. (F) Leaf relative water content from WT and OE plants after 10 days, 15 days drought stress and 3 days rewater treatment. (G) Leaf relative water content from WT and OE plants after 20% PEG-6000 treatment for 48 h."

Fig. 3

Changes of H2O2 content and MDA levels in wild plant and transgenic plant line under 20% PEG treatment. (A) H2O2 content of seedlings at these days of the 20% PEG treatment; (B) MDA levels of seedlings at these days of the 20% PEG treatment. Data are shown as mean±SD of three independent measurements."

Fig. 4

Antioxidant enzyme activities in wild plant and transgenic plants after treatment with 20% PEG(A) SOD activity; (B) POD activity. Data are shown as means ±SD of three independent measurements."

Fig. 5

Overexpression of BnA7HSP70 makes Brassica napus hold more water in soil pot without irrigation for 10 days(A) Relative water content (RWC) of wild type and BnA7HSP70 overexpressing seedling (OE2, OE3, OE7, and OE8) leaves under drought condition. (B) Chlorophyll a and b concentrations were calculated as described in the materials and methods 1.5.3 and combined to give the total chlorophyll concentration, each of which is a mean of the samples taken from 6-8 leaf disks in each pool. Control: the wild and OE plants grew in the irrigated condition; Stress: four-week old wild and OE plants were subjected to progressive drought for seven days."

Fig. 6

BnA7HSP70 overexpression delays drought-induced leaf senescence in OE lines confronted with stress Drought was induced in wild type (WT) and OE transgenic plants (OE2, OE3, OE7, and OE8) at four-week old stage by withholding irrigation for 10 days. Control: normally irrigated plants. Stress: drought-stressed plants. Values are given as mean SD from three replicates. The experiment of senescence-associated genes BnLSC45 and BnLSC222 was induced by drought treatment. Total RNA was isolated from irrigated and drought-stressed mature leaves of wild plants and OE lines, and gene induction was monitored by quantitative RT-PCR using gene-specific primers."

Fig. 7

BnA7HSP70 overexpression increases resistance against tunicamycin (TUN)-induced cell death(A) Seeds and seedlings from overexpressed plants (OE) and untransformed wild-type (WT) plants were exposed to 2.5 µg mL-1 or 5.0 µg mL-1 tunicamycin. (B)-(C) Seedlings were monitored for the development of chlorosis and necrotic lesions, and cell viability were measured by the Evans blue dye method. Abs (600 nm) reflects the dead cell content. The values represent the average of three replicates (±SD)."

Fig. 8

Gene expression analysis of senescence and cell death-associated genes in wild type and OE plants under tunicamycin treatment Total RNA were isolated from wild type and transgenic plant leaves at 0, 24, 48, and 72 h of treatment, and the endogenous BnA7HSP70, BnCNX1, BnBiP3, and BnNRP in wild type and OE transgenic plants treatment with tunicamycin and the control DMSO were monitored by qRT-PCR. The bars indicate the confidence interval (P < 0.05, n = 3). BnCNX1, BnBiP3, and BnNRP are ER stress markers."

Fig. 9

Model for BnA7HSP70 expression on the mobilization of bZIP28 and upregulation of UPR genes(A) In response to the stress, BnA7HSP70 is competed away by the accumulation of misfolded proteins and bZIP28 is proteolytically activated by Golgi-localized S1P or S2P to release bZIP28n, which relocates to the nucleus where it upregulates stress genes including BnBiP3 and BnCNX1; (B) When BnA7HSP70 is overexpressed, accumulated BnA7HSP70 is enough for association with bZIP28 and misfolded proteins. As a result, bZIP28 is detained in the ER even under stress conditions."

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