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. 2017 May 23;12(5):e0177621.
doi: 10.1371/journal.pone.0177621. eCollection 2017.

Expression patterns of members of the ethylene signaling-related gene families in response to dehydration stresses in cassava

Meng Yun Ren  1 , Ren Jun Feng  2 , Hou Rui Shi  1 , Li Fang Lu  3 , Tian Yan Yun  1 , Ming Peng  2 , Xiao Guan  4 , Heng Zhang  1 , Jing Yi Wang  2 , Xi Yan Zhang  2 , Cheng Liang Li  1 , Yan Jun Chen  1 , Peng He  5 , Yin Dong Zhang  1 , Jiang Hui Xie  2
Affiliations

Expression patterns of members of the ethylene signaling-related gene families in response to dehydration stresses in cassava

Meng Yun Ren et al. PLoS One. .

Abstract

Drought is the one of the most important environment stresses that restricts crop yield worldwide. Cassava (Manihot esculenta Crantz) is an important food and energy crop that has many desirable traits such as drought, heat and low nutrients tolerance. However, the mechanisms underlying drought tolerance in cassava are unclear. Ethylene signaling pathway, from the upstream receptors to the downstream transcription factors, plays important roles in environmental stress responses during plant growth and development. In this study, we used bioinformatics approaches to identify and characterize candidate Manihot esculenta ethylene receptor genes and transcription factor genes. Using computational methods, we localized these genes on cassava chromosomes, constructed phylogenetic trees and identified stress-responsive cis-elements within their 5' upstream regions. Additionally, we measured the trehalose and proline contents in cassava fresh leaves after drought, osmotic, and salt stress treatments, and then it was found that the regulation patterns of contents of proline and trehalose in response to various dehydration stresses were differential, or even the opposite, which shows that plant may take different coping strategies to deal with different stresses, when stresses come. Furthermore, expression profiles of these genes in different organs and tissues under non-stress and abiotic stress were investigated through quantitative real-time PCR (qRT-PCR) analyses in cassava. Expression profiles exhibited clear differences among different tissues under non-stress and various dehydration stress conditions. We found that the leaf and tuberous root tissues had the greatest and least responses, respectively, to drought stress through the ethylene signaling pathway in cassava. Moreover, tuber and root tissues had the greatest and least reponses to osmotic and salt stresses through ethylene signaling in cassava, respectively. These results show that these plant tissues had differential expression levels of genes involved in ethylene signaling in response to the stresses tested. Moreover, after several gene duplication events, the spatiotemporally differential expression pattern of homologous genes in response to abiotic and biotic stresses may imply their functional diversity as a mechanism for adapting to the environment. Our data provide a framework for further research on the molecular mechanisms of cassava resistance to drought stress and provide a foundation for breeding drought-resistant new cultivars.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Phylogenetic trees of ethylene receptors and transcription factors in cassava (Manihot esculenta, Me) and Arabidopsis (Arabidopsis thaliana, At).
The phylogenetic trees were generated using MEGA5.0 program with the neighbor-joining method. Bootstrap values from 1000 replicates are indicated at each branch. I, phylogenetic tree of ERFs that clustered into four distinct groups (A, B, C, and D groups); II, phylogenetic tree of ethylene receptors that clustered into two distinct groups (A and B groups); III, phylogenetic tree of EILs that clustered into two distinct groups (A and B groups).
Fig 2
Fig 2. Distribution of ethylene receptor and transcription factor genes on cassava chromosomes.
The chromosome numbers are indicated at the top of each bar. The gene names are on the left side of each chromosome according to the approximate physical locations (right side) of these genes. The triangles indicate the direction of transcription.
Fig 3
Fig 3. Expression profiles of ethylene receptor genes and transcription factor genes in different cassava tissues (stem, leaf, root, and tuber).
The threshold cycle (Ct) values of target and MeActin genes were obtained through qRT-PCR. The expression values for all selected genes was quantified using the 2–ΔCt method, where ΔCt = Ct target gene − Ct actin gene [37]. The Ct of the formula represent mean Ct values of three repeats from three independent qRT-PCR assays. The heat map was generated based on the log2 (expression values+1) with MeV software. Red indicates high expression and white indicates low expression.
Fig 4
Fig 4. Expression profiles of ethylene receptor genes in cassava leaves after drought, osmotic, or salt treatment.
The threshold cycle (Ct) values of target and MeActin genes were obtained through qRT-PCR. The relative expression values for all selected genes was quantified using the 2-ΔΔCt method, where ΔΔCt = (Ct target gene − Ct MeActin gene) treatment − (Ct target gene − Ct MeActin gene) control [37]. Ct of the formula represent mean Ct values of three repeats from three independent qRT-PCR assays. The heat map was generated based on the log2 (relative expression values) with MeV software. Red indicates high expression and white indicates low expression. D, drought stress; O, osmotic stress; S, salt stress. Asterisk (*) on the right corner of number indicate a significant difference at P < 0.05 (Student’s t-test) and absolute log2 (relative expression values) > 1 compared with non-treated control.
Fig 5
Fig 5. Expression profiles of ethylene receptor genes in cassava stems after drought, osmotic, or salt treatment.
The threshold cycle (Ct) values of target and MeActin genes were obtained through qRT-PCR. The relative expression values for all selected genes was quantified using the 2-ΔΔCt method, where ΔΔCt = (Ct target gene − Ct MeActin gene) treatment − (Ct target gene − Ct MeActin gene) control [37]. Ct of the formula represent mean Ct values of three repeats from three independent qRT-PCR assays. The heat map was generated based on the log2 (relative expression values) with MeV software. Red indicates high expression and white indicates low expression. D, drought stress; O, osmotic stress; S, salt stress. Asterisk (*) on the right corner of number indicate a significant difference at P < 0.05 (Student’s t-test) and absolute log2 (relative expression values) > 1 compared with non-treated control. NA denote the expression was too weak to detect.
Fig 6
Fig 6. Expression profiles of ethylene receptor genes in cassava roots after drought, osmotic, or salt treatment.
The threshold cycle (Ct) values of target and MeActin genes were obtained through qRT-PCR. The relative expression values for all selected genes was quantified using the 2-ΔΔCt method, where ΔΔCt = (Ct target gene − Ct MeActin gene) treatment − (Ct target gene − Ct MeActin gene) control [37]. Ct of the formula represent mean Ct values of three repeats from three independent qRT-PCR assays. The heat map was generated based on the log2 (relative expression values) with MeV software. Red indicates high expression and white indicates low expression. D, drought stress; O, osmotic stress; S, salt stress. Asterisk (*) on the right corner of number indicate a significant difference at P < 0.05 (Student’s t-test) and absolute log2 (relative expression values) > 1 compared with non-treated control.
Fig 7
Fig 7. Expression profiles of ethylene receptor genes in cassava tuberous roots after drought, osmotic, or salt treatment.
The threshold cycle (Ct) values of target and MeActin genes were obtained through qRT-PCR. The relative expression values for all selected genes was quantified using the 2-ΔΔCt method, where ΔΔCt = (Ct target gene − Ct MeActin gene) treatment − (Ct target gene − Ct MeActin gene) control [37]. Ct of the formula represent mean Ct values of three repeats from three independent qRT-PCR assays. The heat map was generated based on the log2 (relative expression values) with MeV software. Red indicates high expression and white indicates low expression. D, drought stress; O, osmotic stress; S, salt stress. Asterisk (*) on the right corner of number indicate a significant difference at P < 0.05 (Student’s t-test) and absolute log2 (relative expression values) > 1 compared with non-treated control. NA denote the expression too weak to detect.
Fig 8
Fig 8. Trehalose and proline concentrations in cassava fresh leaves under drought, osmotic, or salt stress condition.
Y-axes denote the concentration (error bars indicate standard deviation of three repeats from three independent assays, p < 0.05). A: The concentration of proline after salt and osmotic treatment; B: The concentration of proline after drought treatment; C: The concentration of trehalose after salt and osmotic treatment; D: The concentration of trehalose after drought treatment.
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Grants and funding

This study was supported by the National Nature Science Foundation of China (31201603, 31501043) to RJF; the National High Technology Research and Development Program of China (863 Programme; no. 2012AA101204-2) to MP; the Major Science and Technology program of Hainan (ZDZX2013023-1) to MP; the Hainan Natural Science Foundation (312050, 312051) to RJF and JYW; and the Graduate Outstanding Dissertation Cultivation Plan of Hainan University (15) to MYR.