Effect of Temperature and ABA Exposure on LlaNAC Expression in Transformed Tobacco Plants

Plant Material and Growth Conditions

Nicotiana tabacum (tobacco) plants over-expressing LlaNAC gene, and co-transformed nptII gene, (four transgenic lines-NC2, NC7b, NC10 and NC18 in generations T2) along with wild-type (WT) plants were maintained in containment under controlled conditions of temperature (25 ± 2°C) and light (16/8 h photoperiod), and grown in soilrite. The plants were nourished with MS basal medium twice a week, and were watered (distilled water) on daily basis. Each individual plant was tested for its genetic stability based on herbicide tolerance assay (150 ppm paromomycin) following PCR assay with an initial denaturation at 94°C for 5 min., followed by 30 cycles of 94°C for 30 s and 55°C for 30 s and 72°C for 1.5 min. and a final extension of 10 min. at 72°C. Amplicons thus obtained was run on 1% agarose gel to check the presence or absence of LlaNAC gene. For whole sets of experiments, six technical replicates were used. The native growth conditions, i.e., temperature (25 ± 2°C) and carbon dioxide (400 ppm) were considered as the control conditions.

Stress Treatments and Gene Expression Analysis

Plants of 10 week old were provided with 10 µM and 50 µM of ABA (Sinha et al. 2014). Regularly watered plants under optimum conditions of temperature (25 ± 2°C) and light (16/8 h photoperiod) for 1 week was considered as control. Heat stress was induced by exposing the 10 week old plants to elevated temperature of 40oC and light (16/8 h photoperiod) in plant growth chamber for 24 h following Pospisilova et al. (2011) [14], Tan et al. (2011) [15], Mackova et al. (2013) [16]. Plants under optimum conditions of temperature (25 ± 2°C) and light (16/8 h photoperiod) for 24 h were considered as control for both the extremes in temperature stress. Expressions of LlaNAC gene was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Total RNA was isolated from young leaf tissues of control and heat exposed (40°C)/ ABA treated (10 and 50 µM). Equal amount (1.0 µg) of the RNA quantified using Qubit fluorometer (Invitrogen, USA) was used for first-strand cDNA synthesis using QuantiTect Reverse Transcription kit (Qiagen, Germany) following manufacturer’s protocol. Quantitative polymerase chain reaction (qPCR) was performed using SYBR® Green JumpStartTM Taq ReadyMixTM (Sigma, USA) kit according to the manufacturer’s instructions. Normalization reaction was carried out with an internal control actin gene using forward (5’-AGGGTTTGCTGGAGATGATG-3’) and reverse (5’-ACCACGTTTG GATTGAGCTT-3’) primers. The PCR amplification was carried out using the primer pair LlaNAC-TqF (5’-ACA GTG GTA AAC CTC CAA AAG G-3’) and LlaNAC-TqR (5’-CGA AGA GAG TTC TTG TTG ACG A- 3’) at an initial denaturation at 94°C for 10 min., followed by 40 cycles of 94°C for 30 s and 55°C for 30 s and 72°C for 30 s. Experiments were performed in triplicate for each set of primers. The transcript was quantified based on ΔΔCt method [17] and presented as fold change over control.

Expression of LlaNAC Transcript under Heat Stress

Heat stress poses a serious threat to global crop production. Plants cope with adverse temperature stress by altering molecular mechanisms and elevated antioxidants activities that decrease levels of stress inducible reactive oxygen species (ROS). Induction of NAC transcription factors in response to stresses activates expression of various stress inducible genes thereby participating in improving stress tolerance [18]. In the present study, the expression of LlaNAC gene over control was noteworthy upon heat exposure to 40°C. Though, the amount of transcript abundance was varied among different lines. NAC Transcript expression in transgenic lines (NC10, NC7b, NC18, NC2) when exposed to temperature stress of 40°C was found significantly increased with a maximum of 1.9 fold in NC7b line, followed by an elevation of 1.4 fold in NC10 line and 1.3 fold in NC18 line over the control (Figure. 2).

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Therefore, we speculated that the rapid increases in LlaNAC transcript abundance might be associated with heat tolerance in transgenic plants. However, the transcript accumulation declined in case of NC2 when compared to the control plants. Similarly, Guo et al. [19] reported up-regulation of TaNAC2L, promotes heat tolerance in transgenic arabidopsis, Han et al. [20] reported over-expression of NAC transcription factors CiNAC4 and CiNAC3 confers salinity tolerance in Arabidopsis, Hong et al. [21] reported the over-expression of ONAC022 for drought and salt tolerance in rice. Plants produce antioxidant enzymes like glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) to cope up with the stress induced by the increased levels of ROS. Further, we noticed elevated antioxidant enzyme activities ranging between 1.5 to 3 fold (data not shown) indicating up-regulation of LlaNAC improves heat tolerance in transgenic plants.

Differential Expression of LlaNAC Transcript by ABA Treatments

ABA is an important signaling molecule in various stresses and often considered as the secondary messenger [19]. As an important phytohormone, ABA controls various processes throughout the life cycle of plants, especially in response to external environmental stimuli [9, 18]. Several NAC genes are involved in stress responses in an ABA-dependent manner [22], while Fang et al. [23] reported SNAC3 confers heat and drought tolerance in ABA independent manner. The inducibility by ABA was clear from the gene expression pattern at both the concentrations. Transgenic lines NC7b, NC18 and NC10 showed up-regulation of NAC gene at 10 µM ABA. NAC transcript accumulation of 1.45 fold was observed in line NC7b, followed by fold of 1.37 in case of lines NC18 and NC10 over the control and again the expression of NC2 line was found repressed (Figure 3). This indicates over-expression of LlaNAC reduced ABA sensitivity at this concentration and is involved in ABA signaling. However, 50 µM ABA leads to down-regulation of NAC expression in all transgenic lines (Figure 3).

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Our results showed that plants over-expressing LlaNAC exhibited hypersensitivity to higher dose of ABA (50 µM) that might be a due to lower transpiration rates and faster stomatal closure in LlaNAC over-expression plants, leading to lower water loss and enhanced tolerance. Similarly, Huang et al. [24] reported TaNAC29-over expressing arabidopsis plants exhibit ABA-hypersensitive response and its expression confers salt and drought tolerance. A relationship between ABA-hypersensitivity and abiotic stress tolerance was established. Upon treatment to 10 µM ABA, elevated levels of GR (2.6 fold) and SOD (1.5 fold) activities were seen between transgenic and WT (data not shown). Interestingly, treatment of 50 µM ABA caused no appreciable increase among transgenic lines. These results indicate LlaNAC transgenic lines exhibited ABA hypersensitivity and were positively regulated by ABA at higher concentrations and provide evidence that LlaNAC participates in the ABA

signal pathway, and plays important roles in stress responses and developmental processes.