Figure 1.
Molecular characterization and phenotype of WsSGTL1 over expressing transgenic Arabidopsis plants.
PCR analysis of three positive transgenic lines of A. thaliana by Gene Specific Primers in T3 generation (amplicon size 2.1 Kb) (A). Three transgenic lines were confirmed by Southern analysis. From Rt to Lt, Lane 1-WT, Lane 2-L1, Lane 3-L2 and Lane 4-L3, were selected for all the experiments (The probes were 700 bp and designed from the 5′ unconserved region of the WsSGTL1) (B). Semi quantitative RT-PCR analysis of two-week-old WT and independent 35S-WsSGTL1 transgenic lines and morphological comparisons of three-week-old WT and WsSGTL1 over-expressing lines under normal growth conditions (C).
Figure 2.
Structural comparison between WsSGTL1 and AtSGT protein.
Three-dimensional model of the WsSGTL1 and AtSGT protein as constructed by Phyre2 server using the backbone ‘C3hbjA’. 3D model of WsSGTL1 with sugar binding domain and sterol binding domain (A). 3D model of AtSGT with sugar binding domain and sterol binding domain highly similar to WsSGTL1 protein structure (B). Structural similarity by superimposition of WsSGTL1 and AtSGT (1.423 Å) (C).
Figure 3.
Phenotypic difference between WT (Col-0) plants and WsSGTL1 overexpression lines of A. thaliana.
Comparison of shoot, root and leaf growth between WsSGTL1 lines (L1, L2 and L3) and WT plants grown on MS medium (A). With 50 mM NaCl (B). With 100 mM NaCl (C). With 150 mM NaCl (D). photographs were taken after 14 days of germination.
Figure 4.
Phenotypes and salt tolerance of the transgenic plants.
Salt stressed 3-weeks-old soil grown plants, irrigated with the indicated NaCl solutions every second day up to 14 days. Phenotypes of plants after 14 d of treatment (A). Dry weight of whole plants measured after 14 days of salt stress. Values are mean ± SE, n = 10, (*) for P≤0.05, (**) for P≤0.01, (***) for P≤0.001 or 0.005, significantly different from the control (t-test) (B). Two-weeks-old seedlings of WT and transgenic lines were used for RNA extraction. To provide salt stress, seedlings were treated with 100 mM NaCl for 24 h before RNA isolation. The transcript level of two stress genes was determined by RT-PCR analyses. The stress genes used for the tests were late embryogenesis abundant proteins LEA4-5 and Salt overlay sensitive gene SOS3 (AF192886) (C).
Table 1.
Survival of plants under salinity conditions in the soil based analysis platform.
Figure 5.
SOD activity and measurement of relative electrical conductivity.
Standard calibration curve for SOD at 595 nm (A). SOD activity analysis in WsSGTL1 transgenic lines of A. thaliana and WT (Col-0) plants. Values are expressed as mean (n = 3); errors bars show the SD for each experiment; (*) P≤0.05 compared to WT (t-test) (B). REC of WsSGTL1 transgenic lines of A. thaliana and WT (Col-0). Values are expressed as mean (n = 3); errors bars show SD.
Table 2.
SOD activity in WT and transgenic lines of A. thaliana.
Figure 6.
Phenotypes and heat tolerance of the transgenic plants.
Thermotolerant phenotypes of WT and overexpression lines of A.thaliana, showing heat sensitivity at 7-d-old seedlings stage. Seedlings were grown on agar plates in light for 7 d and heated at 38°C for 90 min, cooled at room temperature for 120 min, and again heated at 42°C for 180 min (acquired thermotolerance). Percentage of survival of plants in relation to WT control plants on the same plate was determined 5 d after heat stress (A–B). Three-weeks-old soil grown plants exposed directly to 42°C for 60 min (basal thermotolerance). Photograph was taken after 7 day survival of the plants (C). Heat-induced oxidative damage in WT as compared to overexpression lines with decreased thermotolerance. Plants were heat treated as described in Figure (A), and after 2 days of recovery, seedlings were harvested and stored in liquid nitrogen until the assay was performed. The MDA level determined from the overexpression lines of A.thaliana in relation to WT control on each plate was determined. Values are expressed as mean (n = 3); errors bars show the SD for each experiment. (*) P≤0.05 compared to WT (t-test) (D). Two-weeks-old seedlings of WT and all transgenic lines were used for RNA extraction. For heat stress, seedlings were kept at 42°C for 4 h before RNA isolation. The transcript level of two stress genes was determined by RT-PCR analyses. The stress genes used for the tests were Hsp70, Hsp90 (E).
Figure 7.
Phenotypes of 14-d-old seedlings of transgenic plants showing heat tolerance.
Thermotolerant phenotypes of WT and overexpression lines of A.thaliana, showing heat sensitivity at 14-d-old seedling stage. Seedlings were grown on agar plates in light for 14 d and heated to 45°C for 180 min (basal thermotolerance). Photograph was taken after 7 day survival of plants before and after heat stress. (A) WT plants. (B) Transgenic plants.
Table 3.
Survival of plants as determined under heat stress and cold stress in the plate based early analysis platform.
Figure 8.
Phenotypes and cold tolerance of the transgenic plants.
Cold stress was imposed on after 10–12 days of growth. Plates can be transferred directly to a freezing chamber set at −1±0.1°C in the dark to check the basal cold tolerance (NA). For cold acclimated (CA), the plates were first transferred to a cold room set at 4±2°C, under constant light for 7 days. Phenotypes of plants after 2 days score the survival of seedlings visually (A). Freezing tolerance (LT50) of 7-days-old WT and transgenic NA seedlings (B) and CA seedlings (C). Values are expressed as mean (n = 3); error bars show the SD for each experiment; (*) P≤0.05 compared to WT (t-test). Two-weeks-old seedlings of WT and transgenic lines were used for RNA extraction. For cold stress, seedlings were treated at 4°C for 24 h before RNA isolation. The transcript level of two stress genes was determined by RT-PCR analyses. The stress genes used for the tests were RD29a, RD29b (D).
Figure 9.
Chlorophyll Imaging Fluorescence measurements.
Fv/Fm values in WT and WsSGTL1transgenic lines of A.thaliana after normal growth condition of WT (A). Normal growth condition of transgenic line of WsSGTL1 (B). Salt treatments of WT and transgenic lines of 50 mM (C). Salt treatments of WT and Transgenic lines of 100 mM NaCl (D). Heat treatment of (42°C) in WT (E). Heat treatment of (42°C) in transgenic lines of A. thaliana (F). Cold treatment (4°C) in WT (G). Cold treatment of (4°C) in transgenic lines of A. thaliana (H). Salt treatment started after shifting the 14 days seedling plants into the pot for three weeks and all image analysis was done after three week potted plants.
Figure 10.
Analysis of sterols in WT and WsSGTL1 transgenic Arabidopsis lines by HPLC, TLC and enzyme assay.
Comparative analysis of sterols in transgenic Arabidopsis lines expressing WsSGTL1 under the control of the cauliflower mosaic virus 35S promoter. Contents of campestrol, stigmasterol and sitosterol in the leaves of WT and different transgenic lines (A). HPLC purified plant extracts spotted on TLC plate (Lane 1–10) Lane 1 & 4: WsSGTL1 Transgenic plants; Lane 7: Transgenic plant after salt stress (100 mM for 24 h); Lane 2,3,5,6,8: Wild type plants; Lane 9 & 10: Standards Black arrows indicate sterols whereas green arrows indicate sterol glycosides; red arrows indicate standards (B). The crude enzyme activity was determined as cpm or counts per minute and expressed as mCi mmol-1. It was observed that in transgenic plants the enzyme activity was about 5500 mCi mmol-1 which increased to about 7500 mCi mmol-1 in stress conditions as done by radiolabelled enzyme assay (C).
Table 4.
Analysis of different cis-acting regulatory elements in the promoter of WsSGTL1 using PLACE software.