NtLTP4, a lipid transfer protein that enhances salt and drought stresses tolerance in Nicotiana tabacum

Abstract

Lipid transfer proteins (LTPs), a class of small, ubiquitous proteins, play critical roles in various environmental stresses. However, their precise biological functions remain unknown. Here we isolated an extracellular matrix-localised LTP, NtLTP4, from Nicotiana tabacum. The overexpression of NtLTP4 in N. tabacum enhanced resistance to salt and drought stresses. Upon exposure to high salinity, NtLTP4-overexpressing lines (OE lines) accumulated low Na⁺ levels. Salt-responsive genes, including Na⁺/H⁺ exchangers (NHX1) and high-affinity K⁺ transporter1 (HKT1), were dramatically higher in OE lines than in wild-type lines. NtLTP4 might regulate transcription levels of NHX1 and HKT1 to alleviate the toxicity of Na⁺. Interestingly, OE lines enhanced the tolerance of N. tabacum to drought stress by reducing the transpiration rate. Moreover, NtLTP4 could increase reactive oxygen species (ROS)-scavenging enzyme activity and expression levels to scavenge excess ROS under drought and high salinity conditions. We used a two-hybrid yeast system and screened seven putative proteins that interact with NtLTP4 in tobacco. An MAPK member, wound-induced protein kinase, was confirmed to interact with NtLTP4 via co-immunoprecipitation and a firefly luciferase complementation imaging assay. Taken together, this is the first functional analysis of NtLTP4, and proves that NtLTP4 positively regulates salt and drought stresses in N. tabacum.

Figure 1

Characterisation of NtLTP4. (a) Amino acid sequence alignment of N. tabacum NtLTP4 with other LTPs. The sequence of NtLTP4 was 42% identity with the LTP of Arabidopsis thaliana, 55% identity with T. durum, 47% identity with Zea mays and 52% identity with Hordeum vulgare. At, A. thaliana (At5g59310); Td, T. durum (ABO28527.1); Zm, Z. mays (NP_001105392.1); Hv, H. vulgare (CAA85484.1). The conserved cysteine residues were highlighted with asterisks. Conserved and semi-conserved amino acid residues were represented by different colours. The consensus pentapeptides were highlighted in the red box. (b) Amino acid sequence and ribbon model of NtLTP4 structure showing the location of four pairs of disulfide bonds indicated by connecting lines. 3-D structural model of NtLTP4 predicted by SWISS-MODLE. (c) Predicted signal peptide of NtLTP4 (http://www.cbs.dtu.dk/services/SignalP/). (d) Phylogenetic tree of N. tabacum NtLTP4, highlighted by red circle. Nt, N. tabacum (AB625593.1, AB518680.1, AB625594.1); Td, T. durum (JF799976.1); Ta, Triticum aestivum (AJ852557.1, AJ852549.1, AJ852552.1); Hv, H. vulgare (CAA85484, CAA42832); Os, Oryza sativa (AY466108.1, AY466109.1); Zm, Z. mays (ACG30536.1); Si, Setaria italica (LN810550.1); At, A. thaliana (At2g38540, At5g59320, At5g59310, etc.); Ca, C. annuum (AF118131.1); Ng, Nicotiana glauca (AF151214.1); Sb, Sorghum bicolor (X71667.1, X71668.1, XP_002458765.1).

Figure 2

Expression pattern of NtLTP4. (a) Tissue-specific expressions of NtLTP4 in roots, leaves and stems were quantified by qRT-PCR. (b) Total RNA extracted from the whole plants of 14-day-old N. tabacum without treatment and used for qRT-PCR. (c–i) Total RNA extracted from the whole plants of 14-day-old N. tabacum exposed to 200 mM NaCl, 200 mM mannitol, 100 μM ABA, wounding, 5 μM SA, 500 μM MeJA and R. solanacearum were used for qRT-PCR, respectively. Lanes represented the various durations of treatments, the internal control gene was Actin. Experiment was repeated three times. Error bars indicated SEM. One-way ANOVA (Duncan’s multiple range test) was performed, and statistical significant differences were indicated by different lowercase letters (P < 0.01).

Figure 3

Subcellular localization analysis of the NtLTP4 in N. benthamiana leaves and onion epidermal cells. (a) Schematic illustration of the recombinant vector (35S::NtLTP4-GFP) and the control construct (35S::GFP). (b) Transient expression of NtLTP4-GFP and GFP in N. benthamiana leaves. Bars = 20 μm. (c) Transient expression of NtLTP4-GFP and GFP constructs in onion epidermal cells. GFP fluorescence visualised using fluorescence microscopy. Bar = 20 μm. (d) Images showing the magnified regions were indicated by red boxes in (c). Bar = 5 μm. Ten independent materials were analysed. The experiment was repeated with three biological replicates.

Figure 4

Overexpression of NtLTP4 in transgenic plants enhanced salt tolerance. (a) Seeds sown on MS medium containing various concentrations of NaCl. Bar = 1.5 cm. (b) Germination rates of WT and OE lines with or without 200 mM NaCl treatment. (c) Seedling phenotype of 14-day-old WT and OE lines with or without salt stress treatment. Bar = 1 cm. (d) Root length and fresh weight of seedlings shown in (c). (e) Phenotype and survival rates of 8-week-old WT and OE plants grew in soil irrigated with water and 250 mM NaCl solution for 1 month. Bar = 2 cm. (f) Representative phenotypes of leaf disks from detached leaves of WT and OE plants with MS supplied with or without 800 mM NaCl. Bar = 1 cm. (g) Quantification of chlorophyll contents in different plants. Chl.a = chlorophyll a. Chl.b = chlorophyll b. Data were mean values of three biological repeats. Error bars indicated SEM. One-way ANOVA (Duncan’s multiple range test) was performed, and statistical significant differences were indicated by different lowercase letters (P < 0.01).

Figure 5

NtLTP4 regulated Na⁺ homeostasis by controlling the expression levels of HKT1 and NHX1. (a) Na⁺ contents in 14-day-old seedling shoots and roots of WT and OE lines treated with MS or 200 mM NaCl for 48 h exhibited by CoroNa™ Green dye. Green-fluorescence detected by confocal microscopy. Ten independent plants used in detection. Bar = 40 μm. (b) Na⁺ content quantitatively analysed by ImageJ. (c) Contents of Na⁺ and K⁺ in shoots and roots of 8-week-old plants irrigated with water or 250 mM NaCl for one month and measured by atomic absorption spectrometry. (d) Relative expression analysis of NHX1 and HKT1 in 14-day-old WT and OE lines treated with MS or 200 mM NaCl for 48 h. Data as mean values of three biological repeats. Error bars indicated SEM. One-way ANOVA (Duncan’s multiple range test) was performed, and statistical significant differences were indicated by different lowercase letters (P < 0.01).

Figure 6

Overexpression of NtLTP4 in transgenic plants enhanced drought tolerance. (a) Seed germination on MS medium containing various concentrations of mannitol. Bar = 1 cm. (b) Germination rates of WT and OE lines in MS medium with or without 200 mM mannitol. (c) Seedling phenotype of 14-day-old WT and OE lines with or without mannitol treatment. Bar = 1 cm. (d) Root length and fresh weight of seedlings shown in (c). (e) Phenotype and survival rate of 8-week-old WT and OE plants grew in soil with or without dehydration treatment for 15 days. Bar = 2 cm. (f) Photosynthetic performances of WT and OE plants with or without drought treatment. Pn: net photosynthetic rate (Pn, μmol CO₂ m⁻²s⁻¹) Gs: stomatal conductance (gs, mmol H₂O m⁻²s⁻¹) E: transpiration rate (E, mmol H₂O m⁻²s⁻¹). Data as mean values of three biological repeats. Error bars indicated SEM. One-way ANOVA (Duncan′s multiple range test) was performed, and statistical significant differences were indicated by different lowercase letters (P < 0.01).

Figure 7

Overexpression of NtLTP4 in transgenic plants decreased the ROS production and oxidative damage under salt and drought treatments. (a) Photographs of NBT staining and O₂⁻ concentrations measured by kit in WT and OE plants after drought and salt treatments. Bar = 2 cm. (b) Photographs of DAB staining and H₂O₂ concentrations measured by kit in WT and OE plants after drought and salt treatments. Bar = 2 cm. (c) MDA contents in WT and OE plants after salt and drought treatments. (d) SOD, POD and CAT activities measured by corresponding kit, respectively. (e) The expression levels of ROS-scavenging or ROS-producing genes APX, CAT, GST, SOD, RbohA and RbohB in WT and OE lines. Data as mean values of three biological repeats. Error bars indicated SEM. One-way ANOVA (Duncan′s multiple range test) was performed, and statistical significant differences were indicated by different lowercase letters (P < 0.01).

Figure 8

Interaction of NtLTP4 with WIPK. (a) Detection of pGBKT7-NtLTP4-Myc bait fusion protein via Western Blot. Lane 1: pGBKT7 empty insert (negative control), lane 2: NtLTP4-pGBKT7-Myc. (b) Yeast two-hybrid screening assay was conducted in yeast. pGBKT7-p53 and pGADT7-T were used as positive control, and pGBKT7-pLam and pGADT7-T were used as negative control. (c) NtLTP4 interacted with WIPK by yeast-two-hybrid. Bait fusion vector and prey fusion vector pGBKT7-NtLTP4/pGADT7-WIPK and pGBKT7-WIPK/pGADT7-NtLTP4 were transformed into the yeast strain Gold Yeast. (d) NtLTP4 interacted with WIPK by LCI assay. Bar = 2 cm, (e) The interaction between NtLTP4 and WIPK was tested via co-immunoprecipitation (Co-IP) assay of transiently overexpressed WIPK-HA and NtLTP4-GFP in N. benthamiana leaves. N. benthamiana leaves that co-expressed GFP and WIPK-HA were used as negative controls.