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Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A ( PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut

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Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A ( PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut

Tata Santosh Rama Bhadra Rao et al. Front Plant Sci.

Abstract

Eukaryotic translational initiation factor 4A belong to family of helicases, involved in multifunctional activities during stress and non-stress conditions. The eIF4A gene was isolated and cloned from semi-arid cereal crop of Pennisetum glaucum. In present study, the PgeIF4A gene was expressed under the regulation of stress inducible Arabidopsis rd29A promoter in groundnut (cv JL-24) with bar as a selectable marker. The de-embryonated cotyledons were infected with Agrobacterium tumefaciens (LBA4404) carrying rd29A:PgeIF4A construct and generated high frequency of multiple shoots in phosphinothricin medium. Twenty- four T0 plants showed integration of both nos-bar and rd29A-PgeIF4A gene cassettes in genome with expected amplification products of 429 and 654 bps, respectively. Transgene copy number integration was observed in five T0 transgenic plants through Southern blot analysis. Predicted Mendelian ratio of segregation (3:1) was noted in transgenic plants at T1 generation. The T2 homozygous lines (L1-5, L8-2, and L16-2) expressing PgeIF4A gene were exhibited superior growth performance with respect to phenotypic parameters like shoot length, tap root length, and lateral root formation under simulated drought and salinity stresses compared to the wild type. In addition, the chlorophyll retention was found to be higher in these plants compared to the control plants. The quantitative real time-PCR results confirmed higher expression of PgeIF4A gene in L1-5, L8-3, and L16-2 plants imposed with drought/salt stress. Further, the salt stress tolerance was associated with increase in oxidative stress markers, such as superoxide dismutase accumulation, reactive oxygen species scavenging, and membrane stability in transgenic plants. Taken together our results confirmed that the PgeIF4A gene expressing transgenic groundnut plants exhibited better adaptation to stress conditions.

Keywords: JL-24; PgeIF4A; de-embryonated cotyledons; nos-bar; rd29A; segregation; transcript analysis.

Figures

Figure 1
Figure 1
Sequence analysis of PgeIF4A gene and protein. (A) Schematic Representation of three motifs including RNA helicase/DEAD box Rec-Q-motif (34–62, indicated as dotted line), DEAD box domain (183–186, in box), and RNA-helicase C-terminal (246–407) in PgeIF4A protein. (B) Predicted three dimensional structure of PgeIF4A protein showing various domains in different colors, blue represented N-terminal region, magenta for C-terminal, red for α-Helices and green for β-sheets. DEAD box region highlighted in red color circle which harbor Asp-183 in β-5, Glu-184 in linked region and Ala-185, Asp-186 presented in α-9 regions. (C) Phylogenetic tree constructed based on deduced amino acid sequences of various eIF4A from closely related species. Protein names, accession numbers, and species names were indicated at each branch. The phylogenetic tree generated using Neighbor Joining (NJ) method and viewed using MEGA4 software.
Figure 2
Figure 2
Schematic representation of T-DNA region of recombinant plant transformation vector pGreen0229-rd29A:PgeIF4A:poly A and groundnut transformation. (A) PgeIF4A gene was cloned under stress inducible promoter rd29A and poly A terminator, bar (Bialaphos amino transferase gene) as a selectable marker which driven by nos (nopaline synthase) promoter and nos-terminator. LB, left boarder; RB, right boarder. (B) De-embryonated half cotyledons (DEC) prepared from matured seeds. (C,D) DEC producing calli. (E) Phosphinothricin resistant calli producing shoots. (F–H) Shoot induction and multiple shoot formation. (I) Putative transgenic plants (T0) growing in glass house.
Figure 3
Figure 3
Molecular confirmation of putative transgenic groundnut plants (T0). (A) PCR amplification representing the junction of rd29A-PgeIF4A (654bp). (B) Junction of nos-bar marker gene. M, 1 kb marker; PC, positive control (plasmid DNA); NC, negative control; NT, non-transgenic plant; lanes 1–24, transgenic plant samples. (C,D) Alignment of nucleotide sequences of PCR amplified products Ah_rdeIF) and Ah_BAR from transgenic plants with recombinant vector pGreen0229-PgeIF4A sequence. (E) Depicted picture showing the analysis of Southern blot hybridization. The genomic DNA (20 μg) from control and transformed lines were digested with Xho I, size fractionated on 0.8% agarose gel, transferred to nylon membrane (Hybond N+), hybridized with biotin labeled eIF4A probe and detection of chromogenic signal was mediated through alkaline phosphatase reaction. Lane C, non-transgenic plant; Lane L1, L3, L8, L14, and L16 transgenic (T0) plants.
Figure 4
Figure 4
Transgene segregation analysis. (A) Depicted picture showing PCR amplification of eIF4A gene at T1 generation. L1-1 to L1-10, L8-1 to L8-17, and L16-1-L16-13 represents progeny of L1, L8, and L16 (T0) respectively. (B) Effect of PPT on leaves of groundnut transformants showing resistance to herbicide. C, wild type control leaf bleaching to herbicide; T, transgenic showing resistance to the herbicide.
Figure 5
Figure 5
Characterization of eIF4A groundnut transgenic plants under simulated drought stress conditions. (A) Growth performance of PgeIF4A expressing transgenic plants (T2) and control (C) on plain MS medium. (B) Graphical representation of growth performance transgenic plants (T2) and control (C) on 200 and 300 mM mannitol. (C) Relative shoot length in cm. (D) Tap root length in cm. (E) Number of lateral roots. (F) Chlorophyll retention of transgenic and control plants grown under 0, 200, and 300 mM mannitol medium. All the experiments performed in triplicates and data represented as mean (n = three biological triplicates) using two way ANOVA with LSD and P < 0.005. Different alphabets indicated the significant differences between the treatments. Similar letters denotes non significant.
Figure 6
Figure 6
Characterization of PgeIF4A groundnut transgenic plants under simulated salinity stress conditions. (A) Growth performance of PgeIF4A expressing transgenic plants (T2) and control on 100 and 200 mM NaCl containing medium. (B) Graphical representation of relative shoot length in cm. (C) Tap root length in cm. (D) Number of lateral roots. (E) Chlorophyll retention of transgenic and control plants grown under 0, 100, and 200 mM NaCl medium. All the experiments performed in triplicates and data represented as mean (n = three biological triplicates) by using two way ANOVA with LSD and P < 0.005. Different alphabets indicated the significant differences between the treatments. Similar letters denotes non significant.
Figure 7
Figure 7
Expression of PgeIF4A transcript in transgenic groundnut (T2) lines. (A) Semi quantitative RT-PCR showing transcript expression pattern of PgeIF4A and G6PD (Glucose 6 phosphate 1 dehydrogenase) in control and transgenic lines grown under mannitol (drought) and NaCl (salinity) stress conditions. (B) qRT-PCR expression analysis of transgenic plants under un treated (control) and treated (drought and salinity) conditions. The Ct-values of the samples were normalized with G6PD house keeping gene. Lane C: non-transgenic, Lane: L1-5, L8-3, and L16-2 represented T2 transgenic lines.
Figure 8
Figure 8
Response of transgenic plants expressing PgeIF4A to salinity stress. One month old control and transgenic plants were imposed with salinity stress (250 mM NaCl) for 2 weeks. (A) SOD activity (% inhibition of NBT). (B) Lipid peroxidation (malondialdehyde content). (C) Relative electrolyte leakage percentage. (D) DAB (3,3′-Diaminobenzidine) staining of WT and transgenic groundnut leaves. (E) Control (C) and PgeIF4A expressing transgenics (T) allowed to recovery after salinity treatment. Data represented as mean of ±SE (n = 3) using one way ANOVA. *Denotes significant difference between WT treated and transgenics (L1-5, L8-3, and L16-2) at p < 0.05.

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