Simultaneous Expression of Abiotic Stress Responsive Transcription Factors, <i>AtDREB2A, AtHB7</i> and <i>AtABF3</i> Improves Salinity and Drought Tolerance in Peanut (<i>Arachis hypogaea</i> L.)

<div><p>Drought, salinity and extreme temperatures are the most common abiotic stresses, adversely affecting plant growth and productivity. Exposure of plants to stress activates stress signalling pathways that induce biochemical and physiological changes essential for stress acclimation. Stress tolerance is governed by multiple traits, and importance of a few traits in imparting tolerance has been demonstrated. Under drought, traits linked to water mining and water conservation, water use efficiency and cellular tolerance (CT) to desiccation are considered to be relevant. In this study, an attempt has been made to improve CT in drought hardy crop, peanut (<i>Arachis hypogaea</i> L., <i>cv.</i> TMV2) by co-expressing stress-responsive transcription factors (TFs), <i>AtDREB2A, AtHB7</i> and <i>AtABF3</i>, associated with downstream gene expression. Transgenic plants simultaneously expressing these TFs showed increased tolerance to drought, salinity and oxidative stresses compared to wild type, with an increase in total plant biomass. The transgenic plants exhibited improved membrane and chlorophyll stability due to enhanced reactive oxygen species scavenging and osmotic adjustment by proline synthesis under stress. The improvement in stress tolerance in transgenic lines were associated with induced expression of various CT related genes like <i>AhGlutaredoxin</i>, <i>AhAldehyde reductase</i>, <i>AhSerine threonine kinase</i> like protein, <i>AhRbx1</i>, <i>AhProline amino peptidase</i>, <i>AhHSP70</i>, <i>AhDIP</i> and <i>AhLea4</i>. Taken together the results indicate that co-expression of stress responsive TFs can activate multiple CT pathways, and this strategy can be employed to improve abiotic stress tolerance in crop plants.</p></div

Response of peanut transgenic plants co-expressing <i>AtDREB2A</i>, <i>AtHB7</i> and <i>AtABF3</i> to oxidative stress and ethrel-induced senescence.

<p>For oxidative stress, leaf discs were incubated in methyl viologen (5 µM) overnight and exposed to light (1200 µmol.m⁻².s⁻¹) for 1 h. The effect of stress was assessed by estimating reduction in chlorophyll content (<b>a</b>) and cell membrane stability (CMS) (<b>b</b>). For inducing senescence, leaf discs were incubated in ethrel (1200 ppm) overnight and reduction in chlorophyll content (<b>c</b>) and cell viability (<b>d</b>) was estimated.</p

Response of peanut transgenic plants co-expressing <i>AtDREB2A</i>, <i>AtHB7</i> and <i>AtABF3</i> to salinity stress.

<p>Salinity stress (250 mM, NaCl) was imposed to three weeks old plants for 10 days. Shoot (<b>a</b>) and root (<b>b</b>) phenotypes, reduction in chlorophyll content (<b>c</b>), cell membrane stability (CMS) (<b>d</b>), lipid peroxidation (expressed as MDA content) (<b>e</b>), SOD activity (expressed as percent inhibition in NBT reduction) (<b>f</b>) and total dry matter (TDM) (<b>g</b>) recorded 10 days after stress are presented. The bar represents the mean ± SE of triplicate experiments (student’s t test; *P<0.05 versus wild-type).</p

Performance of selected transgenic lines (L1 & L7) co-expressing <i>AtDREB2A</i>, <i>AtHB7</i> and <i>AtABF3</i> under mannitol-induced stress at seedling stage.

<p>The seedlings were exposed to mannitol (200 mM) for seven days and photographed (<b>a</b>). The seedlings were then allowed to recover from stress for 15 days (<b>b</b>). The relative root growth after stress (<b>c & d</b>) and recovery (<b>e & f</b>) was recorded. The bar represents the mean ± SE (n = 8) (student’s t test; *P<0.05 versus wild-type).</p

Phenotype of <i>AtDREB2A</i>, <i>AtHB7</i> and <i>AtABF3</i> co-expressing peanut transgenic lines under NaCl-induced stress at seedling stage.

<p>The seedlings were exposed to NaCl (200 mM) for seven days and photographed (<b>a</b>). The seedlings were then allowed to recover from stress for 15 days (<b>b</b>). The relative root growth after stress (<b>c</b>) and recovery (<b>d & e</b>) were recorded. The bar represents the mean ± SE (n = 8) (student’s t test; *P<0.05 versus wild-type).</p

Response of peanut transgenic plants co-expressing <i>AtDREB2A</i>, <i>AtHB7</i> and <i>AtABF3</i> to drought stress at vegetative stage.

<p>Plants were gradually exposed to drought stress by controlled irrigation and maintained at 20% field capacity for a week. Phenotype of wild type (WT) and transgenic plants (L1 & L7) recorded after recovery from drought stress (<b>a</b>). The relative water content (RWC, <b>b</b>), cell membrane stability (CMS, <b>c</b>) reduction in chlorophyll content (<b>d</b>), and proline content (<b>e</b>) were assessed at 30% FC. The bar represents the mean ± SE (student’s t test; *P<0.05 versus wild-type).</p

Characterization of peanut transgenic plants co-expressing <i>AtDREB2A</i>, <i>AtHB7</i> and <i>AtABF3</i> under normal growth conditions.

<p>Phenotype of wild-type and peanut transgenic lines (L1 & L7) co-expressing <i>AtDREB2A</i>, <i>AtHB7</i> and <i>AtABF3</i> (<b>a</b>). Net photosynthesis (A), stomatal conductance (gs) and <i>in-vivo</i> activity of PSII (Φ_PSII) of wild type (WT) and transgenic lines (<b>b</b>). qRT-PCR showing the relative expression of <i>AtDREB2A</i> (2A), <i>AtHB7</i> (HB7) and <i>AtABF3</i> (ABF3) in selected transgenic (L1 & L7) lines (<b>c</b>). RT-PCR showing the expression pattern of target genes in transgenic lines, L1 and L7 (<b>d</b>).</p

Expression of <i>AtDREB2A</i>, <i>AtHB7</i> and <i>AtABF3</i> target genes in wild type and transgenic plants under drought stress condition.

<p>The transcript levels of nine downstream genes were determined by RT-PCR in drought stressed wild type (WT) and transgenic lines (L1 & L7). The, eukaryotic elongation factor (<i>ELF-A</i>) was used as internal control. The downstream genes used for expression studies were <i>AhProline amino peptidase</i> like protein; <i>AhRing box protein1</i> (<i>AhRbx1</i>); <i>Late embryogenesis abundant 4</i> (<i>AhLEA4</i>); <i>AhGlutaredoxin</i> like protein; <i>AhAldehyde reductase</i> (<i>AhAR</i>) like protein; <i>AhSerine threonine kinase</i> like protein; <i>Heat shock Protein70</i> (<i>AhHSP70</i>); <i>AhCalmodulin</i> like protein; <i>Dehydration inducible protein</i> (<i>AhDIP</i>).</p

3rd National Conference on Image Processing, Computing, Communication, Networking and Data Analytics

This volume contains contributed articles presented in the conference NCICCNDA 2018, organized by the Department of Computer Science and Engineering, GSSS Institute of Engineering and Technology for Women, Mysore, Karnataka (India) on 28th April 2018