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. 2016 Jul 13;17(7):1077.
doi: 10.3390/ijms17071077.

Interspecies and Intraspecies Analysis of Trehalose Contents and the Biosynthesis Pathway Gene Family Reveals Crucial Roles of Trehalose in Osmotic-Stress Tolerance in Cassava

Bingying Han  1 , Lili Fu  2 , Dan Zhang  3 , Xiuquan He  4 , Qiang Chen  5 , Ming Peng  6 , Jiaming Zhang  7
Affiliations

Interspecies and Intraspecies Analysis of Trehalose Contents and the Biosynthesis Pathway Gene Family Reveals Crucial Roles of Trehalose in Osmotic-Stress Tolerance in Cassava

Bingying Han et al. Int J Mol Sci. .

Abstract

Trehalose is a nonreducing α,α-1,1-disaccharide in a wide range of organisms, and has diverse biological functions that range from serving as an energy source to acting as a protective/signal sugar. However, significant amounts of trehalose have rarely been detected in higher plants, and the function of trehalose in the drought-tolerant crop cassava (Manihot esculenta Crantz) is unclear. We measured soluble sugar concentrations of nine plant species with differing levels of drought tolerance and 41 cassava varieties using high-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD). Significantly high amounts of trehalose were identified in drought-tolerant crops cassava, Jatropha curcas, and castor bean (Ricinus communis). All cassava varieties tested contained high amounts of trehalose, although their concentrations varied from 0.23 to 1.29 mg·g(-1) fresh weight (FW), and the trehalose level was highly correlated with dehydration stress tolerance of detached leaves of the varieties. Moreover, the trehalose concentrations in cassava leaves increased 2.3-5.5 folds in response to osmotic stress simulated by 20% PEG 6000. Through database mining, 24 trehalose pathway genes, including 12 trehalose-6-phosphate synthases (TPS), 10 trehalose-6-phosphate phosphatases (TPP), and two trehalases were identified in cassava. Phylogenetic analysis indicated that there were four cassava TPS genes (MeTPS1-4) that were orthologous to the solely active TPS gene (AtTPS1 and OsTPS1) in Arabidopsis and rice, and a new TPP subfamily was identified in cassava, suggesting that the trehalose biosynthesis activities in cassava had potentially been enhanced in evolutionary history. RNA-seq analysis indicated that MeTPS1 was expressed at constitutionally high level before and after osmotic stress, while other trehalose pathway genes were either up-regulated or down-regulated, which may explain why cassava accumulated high level of trehalose under normal conditions. MeTPS1 was then transformed into tobacco (Nicotiana benthamiana). Results indicated that transgenic tobacco lines accumulated significant level of trehalose and possessed improved drought stress tolerance. In conclusion, cassava accumulated significantly high amount of trehalose under normal conditions due to multiplied trehalose biosynthesis gene families and constant expression of the active MeTPS1 gene. High levels of trehalose subsequently contributed to high drought stress tolerance.

Keywords: HPLC-ELSD; Manihot esculenta; RNA-seq; abiotic stress; drought tolerance; phylogenetic analysis; trehalase.

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Figures

Figure 1
Figure 1
Trehalose concentrations in cassava varieties SC5 and SC124: (A) HPLC chromatograph of soluble sugars in cassava (SC5) leaf; (B) distribution of trehalose in cassava organs (SC5); and (C) circadian oscillations of trehalose contents in leaves of cassava varieties SC124 and SC5. The significance of differences was tested by one-way ANOVA and LSD (Least Significant Difference) test using IBM SPSS Statistics Version 24.0 (IBM Corporation, New York, NY, USA). Different letters above columns indicate significant differences at 1% level of significance.
Figure 2
Figure 2
Trehalose concentrations in leaves of different plant species under normal conditions. The contents were measured with HPLC-ELSD method, and presented in mg·g−1 FW (Fresh Weight). The significance of differences was tested by one-way ANOVA, followed by LSD test. Different letters above columns indicate significant differences at 1% level of significance, as analyzed using IBM SPSS Statistics Version 24.0.
Figure 3
Figure 3
Correlations between soluble sugar contents including: trehalose (A); fructose (B); glucose (C); and sucrose (D) in different cassava varieties and water retaining capacities of their detached leaves. The significance of the correlations was analyzed using IBM SPSS Statistics Version 24.0, and the correlation curves were created with Excel 2007 (Microsoft Inc., Seattle, WA, USA). ** and * indicate significant correlations at 1% and 5% levels of significance, respectively.
Figure 4
Figure 4
Changes of soluble sugar concentrations in different tissues of cassava (SC5) in response to 20% PEG treatment for 12 and 24 h: (A) Trehalose; (B) Fructose; (C) Glucose; and (D) Sucrose. The significance of differences were assayed by one-way ANOVA and LSD test using IBM SPSS Statistics Version 24.0. * and ** above columns indicate significant differences compared to Control (CK, 0 h of PEG treatment) under 5% and 1% levels of significance, respectively.
Figure 5
Figure 5
Phylogenetic classification of trehalose-6-phosphate synthase (TPS, A) and trehalose-6-phosphate phosphatase (TPP, B) proteins. The sequences were aligned with ClustlX2 [41], and phylogenies were built using MEGA 7 [42]. Maximum Likelihood (ML), Neighbor-Joining, and Minimum-Evolution methods were used in the phylogenetic analysis and only the ML trees are shown. The confidence probabilities (multiplied by 100) that the interior branch length is greater than 0, as estimated using the bootstrap test (1000 replicates), are presented at the relevant nodes. Scale bars represent 0.1 residue substitution.
Figure 6
Figure 6
Differential expression of trehalose pathway genes in leaves and roots of cassava in response to 20% PEG treatment. These data came from a previous RNA-seq analysis [6]. The expression levels of trehalose pathway genes in all samples in TPM (Transcripts Per kilobase Million) were compiled with Excel 2007, and normalized using logarithmic method with two as base. The normalized dataset was then exported to HemI toolkit [45], and the figure (Heatmap) was built using the default parameters. The expression levels are represented by different colors as indicated by the scale on the bottom right. FEL, full expanded leaf; BL, bottom leaf; FL, folded leaf; RT, root. FEL0, FEL3, and FEL24 indicate full expanded leaf after 0, 3, and 24 h after PEG treatment, respectively; and the treatment lengths of BL, FL, and RT samples were indicated the same as above.
Figure 7
Figure 7
Functional analysis of MeTPS1 gene by transforming it into tobacco: (A) MeTPS1 gene was inserted in the plant expression vector pCAMBIA2300 under the control of CaMV35S promoter; (B) PCR verification of transgenic tobacco plants (Nicotiana benthamiana) (Lanes: M, molecular weight marker; 1, wild type tobacco; 2, A1; 3, A2; 4, A3; 5, A4; 6, pCAMBIA2300-MeTPS1); (C) RT-PCR analysis of MeTPS1 gene in homozygous transgenic lines (Lanes: M, molecular marker; R, root; S, stem; L, leaf; I, inflorescence; WT, wild-type control); (D) trehalose concentrations in homozygous transgenic lines, where * indicates significant differences at 1% level of significance compared to the relevant organ of wild type (WT); (E) representative transgenic plants and wild-type (WT) after 30 days of water-withheld; and (F) the same plants as in (E) one day after re-watering.
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