RNAi-mediated gene silencing of <i>WsSGTL1</i> in <i>W.somnifera</i> affects growth and glycosylation pattern

<p>Sterol glycosyltransferases (SGTs) belong to family 1 of glycosyltransferases (GTs) and are enzymes responsible for synthesis of sterol–glucosides (SGs) in many organisms. <i>WsSGTL1</i> is a SGT of <i>Withania somnifera</i> that has been found associated with plasma membranes. However its biological function in <i>W.somnifera</i> is largely unknown. In the present study, we have demonstrated through RNAi silencing of <i>WsSGTL1</i> gene that it performs glycosylation of withanolides and sterols resulting in glycowithanolides and glycosylated sterols respectively, and affects the growth and development of transgenic <i>W.somnifera</i>. For this, RNAi construct (pFGC1008-<i>WsSGTL1</i>) was made and genetic transformation was done by <i>Agrobacterium tumefaciens</i>. HPLC analysis depicts the reduction of withanoside V (the glycowithanolide of <i>W.somnifera</i>) and a large increase of withanolides (majorly withaferin A) content. Also, a significant decrease in level of glycosylated sterols has been observed. Hence, the obtained data provides an insight into the biological function of <i>WsSGTL1</i> gene in <i>W.somnifera</i>.</p

Characterization of <i>Arabidopsis</i> sterol glycosyltransferase <i>TTG15/UGT80B1</i> role during freeze and heat stress

<p>Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of <i>TTG15/UGT80B1</i> gene of <i>Arabidopsis thaliana</i> in freeze/thaw and heat shock stress using T-DNA insertional <i>sgt</i> knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, <i>TTG15/UGT80B1</i> knockout mutant and <i>p</i>35<i>S</i>:<i>TTG15/UGT80B1</i> restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, <i>p</i>35S:<i>TTG15/UGT80B1</i> restored plants were more adapted to freeze stress than <i>TTG15/UGT80B1</i> knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both β-sitosterol and sitosterol glycoside quantity was more in Col-0 and <i>p</i>35<i>S</i>:<i>TTG15/UGT80B1</i> restored lines, whereas it was significantly less in <i>TTG15/UGT80B1</i> knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress.</p

Phenotypes and salt tolerance of the transgenic plants.

<p>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 (<b>A</b>)<b>.</b> Dry weight of whole plants measured after 14 days of salt stress. Values are mean ± SE, n = 10, (*) for <i>P≤0.05</i>, (**) for <i>P≤0.01,</i> (***) for <i>P≤0.001</i> or <i>0.005,</i> significantly different from the control (t-test) (<b>B</b>)<b>.</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 <i>LEA4-5</i> and Salt overlay sensitive gene <i>SOS3</i> (AF192886) (<b>C</b>)<b>.</b></p

Phenotypes and heat tolerance of the transgenic plants.

<p>Thermotolerant phenotypes of WT and overexpression lines of <i>A.thaliana,</i> 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 (<b>A–B</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 (<b>C</b>). Heat-induced oxidative damage in WT as compared to overexpression lines with decreased thermotolerance. Plants were heat treated as described in Figure (<b>A)</b>, 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 <i>A.thaliana</i> 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. (*) <i>P≤0.05</i> compared to WT (t-test) (<b>D</b>)<b>.</b> 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 <i>Hsp70, Hsp90</i> (<b>E</b>).</p

Chlorophyll Imaging Fluorescence measurements.

<p>Fv/Fm values in WT and <i>WsSGTL1</i>transgenic lines of <i>A.thaliana</i> after normal growth condition of WT (<b>A</b>)<b>.</b> Normal growth condition of transgenic line of <i>WsSGTL1</i> (<b>B</b>)<b>.</b> Salt treatments of WT and transgenic lines of 50 mM (<b>C</b>)<b>.</b> Salt treatments of WT and Transgenic lines of 100 mM NaCl (<b>D</b>)<b>.</b> Heat treatment of (42°C) in WT (<b>E</b>)<b>.</b> Heat treatment of (42°C) in transgenic lines of <i>A. thaliana</i> (<b>F</b>)<b>.</b> Cold treatment (4°C) in WT (<b>G</b>)<b>.</b> Cold treatment of (4°C) in transgenic lines of <i>A. thaliana</i> (<b>H</b>)<b>.</b> 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.</p

Structural comparison between WsSGTL1 and AtSGT protein.

<p>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 (<b>A</b>). 3D model of AtSGT with sugar binding domain and sterol binding domain highly similar to WsSGTL1 protein structure (<b>B</b>). Structural similarity by superimposition of WsSGTL1 and AtSGT (1.423 Å) (<b>C</b>).</p

SOD activity and measurement of relative electrical conductivity.

<p>Standard calibration curve for SOD at 595 nm <b>(A).</b> SOD activity analysis in <i>WsSGTL1</i> transgenic lines of <i>A. thaliana</i> and WT (Col-0) plants. Values are expressed as mean (n = 3); errors bars show the SD for each experiment; (*) <i>P≤0.05</i> compared to WT (t-test) (<b>B</b>)<b>.</b> REC of <i>WsSGTL1</i> transgenic lines of <i>A. thaliana</i> and WT (Col-0). Values are expressed as mean (n = 3); errors bars show SD.</p

Molecular characterization and phenotype of <i>WsSGTL1</i> over expressing transgenic <i>Arabidopsis</i> plants.

<p>PCR analysis of three positive transgenic lines of <i>A. thaliana</i> by Gene Specific Primers in T₃ generation (amplicon size 2.1 Kb) (<b>A</b>). 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 <i>WsSGTL1</i>) (<b>B</b>). Semi quantitative RT-PCR analysis of two-week-old WT and independent <i>35S-WsSGTL1</i> transgenic lines and morphological comparisons of three-week-old WT and <i>WsSGTL1</i> over-expressing lines under normal growth conditions (<b>C</b>).</p

Analysis of different cis-acting regulatory elements in the promoter of <i>WsSGTL1</i> using PLACE software.

<p>Analysis of different cis-acting regulatory elements in the promoter of <i>WsSGTL1</i> using PLACE software.</p

Survival of plants under salinity conditions in the soil based analysis platform.

<p>Comparison of various growth parameters of WT (untransformed) and <i>WsSGTL1</i> transgenic <i>Arabidopsis</i> plants grown under normal (0 mM), 50 mM, 100 mM and 150 mM NaCl conditions. Salinity stress was imposed on 4-weeks-old soil-grown plants, which were irrigated with the indicated NaCl solutions every second day for 14 d. Data represents the mean and standard error (<b>±</b>SE) (<i>n = </i>35). Rosette diameter was calculated by comparing data from normal and salt treated plants. Similar results were obtained from three independent experiments. Extent of leaf chlorosis was visually rated on a scale percentage. Normal condition showed no chlorosis (data not presented). ‘0’ denotes no survival in case of WT.</p