Overexpression of EcbHLH57 Transcription Factor from Eleusine coracana L. in Tobacco Confers Tolerance to Salt, Oxidative and Drought Stress

Abstract

Basic helix-loop-helix (bHLH) transcription factors constitute one of the largest families in plants and are known to be involved in various developmental processes and stress tolerance. We report the characterization of a stress responsive bHLH transcription factor from stress adapted species finger millet which is homologous to OsbHLH57 and designated as EcbHLH57. The full length sequence of EcbHLH57 consisted of 256 amino acids with a conserved bHLH domain followed by leucine repeats. In finger millet, EcbHLH57 transcripts were induced by ABA, NaCl, PEG, methyl viologen (MV) treatments and drought stress. Overexpression of EcbHLH57 in tobacco significantly increased the tolerance to salinity and drought stress with improved root growth. Transgenic plants showed higher photosynthetic rate and stomatal conductance under drought stress that resulted in higher biomass. Under long-term salinity stress, the transgenic plants accumulated higher seed weight/pod and pod number. The transgenic plants were also tolerant to oxidative stress and showed less accumulation of H202 and MDA levels. The overexpression of EcbHLH57 enhanced the expression of stress responsive genes such as LEA14, rd29A, rd29B, SOD, APX, ADH1, HSP70 and also PP2C and hence improved tolerance to diverse stresses.

Fig 1. Expression analysis of EcbHLH57 in finger millet under different stress conditions.

A) Quantitative RT-PCR analysis of EcbHLH57 in finger millet plants subjected to drought stress of 80%, 60%, 40% FC and in seedlings treated with B) ABA (10 μM), C) PEG 6000 (-12 bars), D) NaCl (300 mM) and E) MV (10 μM) at different time points. Total RNA was isolated and cDNA prepared was used for quantitative RT-PCR analysis. Actin was used as internal control. Data represent mean of three replications (n = 3) and bars indicate standard error of mean. The lowercase letters that are different indicate significant difference (Duncan’s multiple range test, P<0.05) between treatment conditions.

Fig 2. Expression and phenotypic analysis of EcbHLH57 expressing tobacco transgenic and wild type plants.

A) qRT-PCR analysis of tobacco transgenic plants overexpressing EcbHLH57. Photograph showing phenotype of wild type and transgenic plants at B) 90 days and C) 120 days after germination. Histogram showing D) Plant height and E) Leaf area of third fully expanded leaf, F) Internode length and G) Stem girth of transgenic and wild type plants. Arrows indicate distance between two internodes. Data represent mean of three replications (n = 3) and bars indicate standard error of mean. The lowercase letters that are different indicate significant difference (Duncan’s multiple range test, P<0.05) between transgenic and wild type plants. WT-wild type, M2, M3 and M4- Transgenic plants.

Fig 3. Leaf anatomical study and microscopic view of epidermal impression of transgenic and wild type plants (40X magnification).

A) Leaf cross-sections observed under microscope. For histological studies, the top third leaf of transgenic and wild type plants was taken and microtome sections were made. Staining was done using eosin (stains nucleus) and hematoxylin (stains cytoplasm). B) Photograph showing abaxial surface of leaves from transgenic and wild type plants. C) Graph depicting stomatal index of transgenic and wild type plants. Data represent mean of three replications (n = 3) and bars indicate standard error of mean. The lowercase letters that are different indicate significant difference (Duncan’s multiple range test, P<0.05) between transgenic and wild type plants. x- xylem, p-phloem, S-spongy parenchyma, P-palisade parenchyma, ad-Adaxial, ab- abaxial.

Fig 4. Response of EcbHLH57 expressing transgenic plants for salinity stress tolerance.

A) Root length and cotyledon greening rate of EcbHLH57 expressing transgenic and wild type seedlings under 100mM NaCl treatment. Graph depicting i) Root length and ii) Cotyledon greening rate of transgenic and wild type plants. B) 30-day-old plants were treated with 300 mM NaCl for 1 week. i) Root growth comparison of transgenic and wild type plants. ii) Per cent reduction in shoot and root fresh weight of plants over water control iii) Percent reduction in chlorophyll content. iv) Electrolyte leakage under NaCl treatment. Data represent mean of three replications (n = 3) and bars indicate standard error. The lowercase letters that are different indicate significant difference (Duncan’s multiple range test, P<0.05) between transgenic and wild type plants exposed to same treatment. WT- wild type. M2, M3 and M4- transgenic lines expressing EcbHLH57.

Fig 5. Response of EcbHLH57 expressing transgenic plants to PEG, NaCl and MV induced stress.

Leaf discs from wild type and transgenic plants were placed on half MS medium supplemented with PEG 6000 (-6 bars), NaCl (200 mM) and MV (2 μM). A) Photograph taken 72 h after exposure to treatment. Leaf discs were analyzed for per cent reduction in chlorophyll content over water control under, B) PEG, C) NaCl and D) MV treatments. Analysis of, E) H₂O₂ content using xylenol orange assay and, F) MDA content using TBARS assay in MV treated leaf discs. Data represent mean of three replications (n = 3) and bars indicate standard error. The lowercase letters that are different indicate significant difference (Duncan’s multiple range test, P<0.05) between transgenic and wild type plants exposed to same treatment. WT- wild type. M2, M3 and M4- transgenic lines expressing EcbHLH57.

Fig 6. Effect of gradual moisture stress on photosynthetic efficiency in EcbHLH57 expressing transgenic plants.

Moisture stress was imposed by gradual reduction of soil moisture status to 60% and 40% FC. 100% FC was maintained as control. Photosynthetic measurements were made at the end of the stress period using IRGA (LiCOR, USA). A) The photosynthesis. B) Stomatal conductance and C) Comparison of dry weight of transgenic and wild type plant at the end of stress period. Data represent mean of three replications (n = 3) and bars indicate standard error. The lowercase letters that are different indicate significant difference (Duncan’s multiple range test, P<0.05) between transgenic and wild type plants exposed to same treatment. WT- wild type. M2, M3 and M4- transgenic lines expressing EcbHLH57.

Fig 7. Expression analysis of downstream genes under drought stress in EcbHLH57 transgenic plants.

Quantitative RT-PCR analysis of a few downstream genes in transgenic and wild type plants under drought stress conditions. Elf- elongation factor was used as internal control. Data represent mean of three replications (n = 3) and bars indicate standard error. The lowercase letters that are different indicate significant difference (Duncan’s multiple range test, P<0.05) between means of analyzed genes.