A Novel Non-Specific Lipid Transfer Protein Gene, CmnsLTP6.9, Enhanced Osmotic and Drought Tolerance by Regulating ROS Scavenging and Remodeling Lipid Profiles in Chinese Chestnut (Castanea mollissima Blume)

Abstract

Chestnut (Castanea mollissima Blume) is an important economic tree owing to its tasty fruit and adaptability to environmental stresses, especially drought. Currently, there is limited information about non-specific lipid transfer protein (nsLTP) genes that respond to abiotic stress in chestnuts. Here, a chestnut nsLTP, named CmnsLTP6.9, was identified and analyzed. The results showed that the CmnsLTP6.9 protein localized in the extracellular matrix had two splicing variants (CmnsLTP6.9L and CmnsLTP6.9S). Compared with CmnsLTP6.9L, CmnsLTP6.9S had an 87 bp deletion in the 5'-terminal. Overexpression of CmnsLTP6.9L in Arabidopsis enhanced tolerance to osmotic and drought stress. Upon exposure to osmotic and drought treatment, CmnsLTP6.9L could increase reactive oxygen species (ROS)-scavenging enzyme activity, alleviating ROS damage. However, CmnsLTP6.9S-overexpressing lines showed no significant differences in phenotype, ROS content, and related enzyme activities compared with the wild type (WT) under osmotic and drought treatment. Moreover, lipid metabolism analysis confirmed that, unlike CmnsLTP6.9S, CmnsLTP6.9L mainly altered and upregulated many fatty acyls and glycerophospholipids, which implied that CmnsLTP6.9L and CmnsLTP6.9S played different roles in lipid transference in the chestnut. Taken together, we analyzed the functions of CmnsLTP6.9L and CmnsLTP6.9S, and demonstrated that CmnsLTP6.9L enhanced drought and osmotic stress tolerance through ROS scavenging and lipid metabolism.

Keywords: CmnsLTP; CmnsLTP6.9; chestnut; drought; osmotic.

Figure 1

Phylogenetic tree of nsLTP proteins from C. mollissima, A. thaliana, and B. napus. Nine nsLTP protein types are marked using different colors. Stars represent the genes of C. mollissima. Blue lines represent the genes of C. mollissima. Green lines represent the genes of B. napus. Red lines represent the genes of A. thaliana. Black dots represent the clades’ support values in the phylogenetic trees.

Figure 2

Gene structure and motif compositions of CmnsLTPs. Left: conserved motif composition of CmnsLTPs. The different colored boxes represent different motifs. The scale bar at the bottom represents 30 aa. Right: intron–exon structure of CmnsLTPs. Yellow boxes represent exons; gray lines represent introns. The scale bar at the bottom represents 1000 bp.

Figure 3

Responsive cis-acting elements predicted in the CmnsLTPs promoters. Different colors represent different responsive elements.

Figure 4

Expression profiling of CmnsLTPs. (A) Expression analysis of CmnsLTPs in various tissues of chestnut. The visualization of the result was achieved using TBtools. Different colored CmnsLTPs represent different expression patterns. The color scale represents the relative signal intensity. M1, M2, M3, and M4 represent male chestnut flowers at four developmental stages. M1, flower bud primordium differentiation stage; M2, perianth formation stage; M3, pollen mother cell and tetrad stage; M4, pollen grain maturation period. F1 represents the female chestnut flower at the ovule differentiation stage; F2, maturation period. (B) Heat map representation of CmnsLTPs under osmotic (0 h, 1 h, 3 h, 6 h, and 24 h) and drought (0 h, 1 h, 3 h, 6 h, and 9 h) stress from the qRT-PCR experiment. The color bar represents the relative signal intensity value.

Figure 5

Protein sequence alignment and subcellular localization analysis. (A) Protein sequence alignment of CmnsLTP6.9S and CmnsLTP6.9L. The sequences in the red boxes are conserved domains. Black highlights represent the highly conserved protein sequence and blue highlights represent the different protein sequence between C. mollissima and A. thaliana. (B) Transient expression of CmnsLTP6.9S-GFP and CmnsLTP6.9L-GFP in onion epidermal cells. Bars = 20 μm.

Figure 6

Overexpression of CmnsLTP6.9L in transgenic plants enhanced for osmotic and drought tolerance. (A) Phenotype of 1-month-old WT and OE plants grown in soil irrigated with water, 250 mM mannitol solution, and without water for 2 weeks. (B) Relative water content. (C) Relative electrolyte leakage. (D) Proline content. (E) MDA content. The data are the mean values of three biological repeats. The error bars indicate the SE. One-way ANOVA (Duncan) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.05).

Figure 7

The overexpression of CmnsLTP6.9L in transgenic plants decreased the ROS content and oxidative damage under osmotic and drought treatment. (A) Photographs of DAB and NBT staining in WT and OE plants under osmotic and drought treatment. (B) H₂O₂ concentrations measured by the kit. (C,D) SOD and POD activities are measured by the corresponding kits, respectively. The error bars indicate the SE. One-way ANOVA (Duncan) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.05).

Figure 8

Statistics on the numbers of DELs. (A) Venn diagram statistics of the comparisons among the different groups. W, WT; L1, CmnsLTP6.9L; L2, CmnsLTP6.9S. (B) Statistical analysis of DELs in the different groups in the different plants. (C) Relative contents of different lipids in the plants. GP, glycerophospholipids; FA, fatty acyls; PR, prenol lipids; SL, saccharolipids; SP, sphingolipids; ST, sterol lipids; PK, polyketides; GL, glycerolipids. The dotted lines represent inter-group intervals and intra-group comparisons. The error bars indicate the SE. One-way ANOVA (Duncan) was performed, and statistically significant differences are indicated by different lowercase letters (p < 0.05).

Figure 9

A potential model of CmnsLTP6.9 regulating plant responses to osmotic and drought stress. Osmotic and drought stress induced the expression of CmnsLTP6.9. After osmotic and drought stress, CmnsLTP6.9-OE increased the activities of POD and SOD in Arabidopsis thaliana, thus scavenging H₂O₂ and O₂⁻. However, CmnsLTP6.9-OE remodeled the lipid content of A. thaliana, thus increasing the content of PI and PC and promoting tolerance to drought and osmotic stress.