Introduction of Arabidopsis’s heat shock factor HsfA1d mitigates adverse effects of heat stress on potato (Solanum tuberosum L.) plant

Abstract

Thermal stress induces a wide array of morphological and physiological changes in potato affecting its development and economic yield. Response to thermal stress in plants is mostly regulated by heat shock factors (hsfs). The current study aimed at improving heat tolerance by transforming potato plant with heat shock factor, HsfA1d, using Agrobacterium. Gateway cloning strategy was adopted for isolation of HsfA1d from Arabidopsis thaliana and cloning into plant expression vector. The target gene was introduced into potato by infecting internodal explants with Agrobacterium strain GV3101 carrying pGWB402Ω-HsfA1d construct. Upon exposure to heat stress, the wild-type plants turned yellowish, whereas no phenotypic effect on transgenic plants was observed. Expression of HsfA1d in transgenic plants was increased by 5.8-fold under thermal stress compared to room temperature. Transgenic plants exhibited 6-fold increase in the expression of downstream HSP70 under thermal stress compared to wild-type plants. Both chlorophyll a and b were significantly decreased in wild-type plants while no such decrease was recorded in transgenic plants under thermal stress. Heat stress was found to have no significant effect on carotenoid pigments of both wild-type and transgenic plants. Significantly lower electrolyte leakage from transgenic plants was witnessed compared to wild type upon exposure to thermal stress. Transgenic plants accumulated significantly higher proline content compared to wild-type plants under heat stress. It is concluded that HsfA1d plays a vital role in plant thermotolerance and hence can be effectively used to enhance the resistance of crop plants against heat stress.

Electronic supplementary material

The online version of this article (10.1007/s12192-019-01043-6) contains supplementary material, which is available to authorized users.

Introduction

Potato, a starchy tuberous crop, is globally consumed as a food and provides raw material to agro-based industries. Potato best grows in frost-free cool seasons and is highly prone to heat stress (Hijmans 2003). The temperature range for the aboveground part of the potato plant is 20–25 °C whereas the optimal range for tuberization 15–20 °C (Van Dam et al. 1996). Elevated temperature induces a variety of changes in potato plants, affecting its development and reducing economic yield. Under thermal stress, the partitioning of photoassimilate to tubers is significantly reduced which greatly affects tuberization potential of the plant (Lafta and Lorenzen, 1995). Extensive denaturation and aggregation of cellular proteins is induced under heat stress, which if not addressed properly may result in cell death. The survival of cell under stress is primarily dependent on structure and function of the intracellular proteins (Wang et al. 2004).

The plants produce heat-shock proteins (HSPs) upon exposure to heat stress. On the basis of their molecular weights, hsps are categorized into five groups; (1) HSP60, (2) HSP70, (3) HSP90, (4) HSP100, and (5) small heat shock proteins (sHSPs) (Datta et al.2017). The principal role of HSPs, during heat stress, is to restrict aggregation of the cellular proteins. Heat shock transcription factors (HSFs) act as main player in regulating expression of the HSPs (Scharf et al.2012). Under thermal stress, HSF is shifted to the nucleus and phosphorylated to form activated trimers. The trimeric HSF binds to promoter sequence of the HSP gene known as heat shock elements (HSEs). Binding of the HSF to HSE results in the generation of HSPs (Lata and Prasad, 2011). The existing cellular proteins and newly synthesized HSPs stabilize the unfolded proteins. Though, 27 StHSFs in the potato genome operating under adverse conditions have been identified (Tang et al.2016), potato is perceived as heat-sensitive crop. On the other hand, Arabidopsis thaliana has been reported to synthesize 21 HSFs (Nover et al.2001). Among these, HsfA1d has been found to involve in acquisition of plant thermotolerance (Hsiang-chin and Yee-yung, 2012)

The nucleotide sequence of HsfA1d isolated from Arabidopsis thaliana has shown least homology (52%) with its homologs in potato genome. To address the negative impact of thermal stress on growth and production of potatoes, research has been focussed on the development of thermotolerant potatoes using different genetic approaches. High degree of sterility and complexity of genome make potato less appealing for conventional breeding (Romano et al.2003). Thus, genetic engineering provides valuable tools for improvement of potato germplasm against the heat stress (Trapero-Mozos et al.2017). In the current study, HsfA1d was isolated from Arabidopsis thaliana and transformed in potato for enhancement of its thermotolerance.

Materials and methods

Preparation of entry clone

Total RNA from leaves of Arabidopsis thaliana (Col-O) was isolated using RNeasy® mini kit (Qiagen, Germany), and followed by treating with DNase-I (Wiame et al.2000). Complementary DNA (cDNA) was then generated from the total RNA using the Super Script Transcriptase III Kit (Life Technologies, CA, USA) and the oligo (dt) primers according to the supplier’s instructions. The cDNA was used as a template and denatured at 94 °C for 2 min before the PCR-based amplification. The AtHsfA1d was amplified with gene-specific primers using the newly synthesized cDNA as template.

The double digestion of the pUC57GW-CmRccdB with the NcoI and XhoI resulted in the formation of linearized vector. The linearized vector (2804 bp) was purified with QIAquick® gel extraction kit (Qiagen, Germany). HsfA1d was infused in the linearized vector (pUC57GW) using In-Fusion® cloning kit (Clontech, USA). Highly competent stellar cells (Clontech, USA) were transformed with the cloning product using heat shock method. Plasmid was extracted from the colonies using QIAprep spin miniprep kit. The restriction enzymes, NcoI and BglII, were used for confirmation of the entry clone. Clustal omega and APE software were used for sequence alignment. HsfA1d was sub-cloned from the entry clone (pUC57GW-HsfA1d) into plant expression vector (pGWB402Ω) using LR clonase (Invitrogen Life Technology, USA). Agrobacterium tumefaciens GV3101 were transformed with pGWB402Ω-HsfA1d following heat shock method.

Agrobacterium-mediated plant transformation

Explant preparation

Potato tubers were grown in soil for 3 weeks. Nodal sections were excised and sterilized with 10% bleach followed by rinsing with autoclaved water. The sterilized nodal parts were grown on hormone-free MS media for 3 weeks to develop in vitro plantlets. Internodal sections of plantlets were used as explants for agro-infection.

Inoculum preparation

The Agrobacterium tumefaciens GV3101 harboring the plasmid pGWB402Ω-HsfA1d were allowed to grow overnight in LB broth containing spectinomycin (50 mg L⁻¹) at 28 °C. The resultant bacterial culture was allowed to grow for another 3 h after the addition of 45 mL fresh LB broth. The culture was centrifuged, and the pellet was washed twice with 20 mL MMA wash buffer. Finally, the inoculum was prepared by re-suspending the pellet in MS media with OD adjusted to 0.6 (OD₆₀₀ = 0.6). Acetosyringone (100 μM) was added just before infecting the explants.

Explant transformation and regeneration

Transgenic potato plants were developed by following the methods of Jung et al. (2005) with some modifications. Potato internodes were infected with the inoculum of Agrobacterium GV3101 for 10 min at 90 rpm and 22 °C. The infected explants were co-cultivated on MS media containing acetosyringone (100 μM) for 3 days in dark. The explants were transferred to MS media with zeatin riboside (ZR) (0.8 mg L⁻¹), 2, 4-D (2.0 mg L⁻¹), kanamycin (100 mg L⁻¹) and grown under 16 h light)/8 h dark at 24 ± 2 °C for 4 weeks. The calli were shifted to MS with ZR (0.8 mg L⁻¹) and GA₃ (2.0 mg L⁻¹) for shoot induction. The shoots were grown on MS containing sucrose (60 mg L⁻¹), kinitin (2.5 mg L⁻¹), and gelrite (2 mg L⁻¹), for 1 month under complete dark to induce in vitro tuberization. The in vitro tubers were grown in autoclaved 2B soil for 3 weeks to produce seedling.

Confirmation of the transgenic plants

Genomic DNA from the putative transgenic plants was isolated using Qiagen DNeasy plant mini kit and amplified through PCR with the gene-specific primers (HsfA1d) following the optimized PCR conditions: 2 min initial denaturation at 95 °C (1 cycle), denaturation at 95 °C for 30 s (30 cycles), annealing at 55 °C for 30 s (30 cycles) and extension at 72 °C for 1.5 min (30 cycles) with a final extension at 72 °C for 5 min. Sequence of the primers used for the detection of transgene (HsfA1d) was as follows:

HsfA1d-F: 5-GCCGCCTTCACCATGGATGTGAGCAAAGTAACCAC-3

HsfA1d-R: 5-CTGGGTCACCCTCGATCAAGGATTTTGCCTTGAGGGATC-3

Heat treatment

The T₁ plants were exposed to heat at 42 °C for 1 week in growth chamber. The effect of heat treatment was observed phenotypically, and samples were collected for the following expression and physiological analysis:

Expression analysis of transgenic plants

Total RNA from transgenic and wild-type plants were extracted as described in Qiagen RNeasy plant mini kit. RNA was first treated with DNase (Wiame et al., 2000) and then converted into first-strand cDNA using Super Script Transcriptase III Kit (Life Technologies, CA, USA) and the oligo (dt) primers. Reaction mixture for expression analysis was prepared by adding, SYBR Green (5 μl), cDNA (2 μl), 0.3 μl each of forward and reverse primers and water (2.4 μl) to a centrifuge tube. The thermal profile applied was consist of initial denaturation 98 °C for 2 min, followed by 42 cycles, with each one having denaturation 98 °C for 10 s, annealing 55 °C for 30 s, extension 72 for 2 min. Finally, 72 °C for 10 min was employed.

Determination of photosynthetic pigments

Lichtenthaler and Wellburn (1983) method was followed for determination of chlorophyll “a”, “b” and total carotenoids. Leaf sample (25 mg) was mixed with magnesium oxide (MgO, 25 mg) followed by the addition of methanol (5 ml) and homogenizing on shaker for 2 h. The resultant extract was exposed to centrifugation for 5 min using 4000 rpm at normal temperature. Absorbance of the supernatant was recorded at three different wave lengths (470 nm, 653 nm, and 666 nm) against a solvent blank using spectrophotometer (UV-2600).

The following formulas were used for calculation of photosynthetic pigments.

Chlorophyll a = 15.65 A₆₆₆–7.340 A₆₅₃

Chlorophyll b = 27.05 A₆₅₃–11.21 A₆₆₆

Total carotenoids = (1000 A₄₇₀–2.860 Chla–129.2 Chlb)/245

Measurement of electrolyte leakage (EL)

The method of Bajji et al. (2002) with slight modifications was used for measurement of EL. Ten pieces (1 cm) each from three different seedlings of both transgenic and wild-type plants were collected, after heat treatment. The leaf samples were washed three times and placed in deionized water (10 mL). The conductivity recorded at the start of rehydration period was denoted as ECi. The tubes containing the leaf segments were kept at normal room temperature for 24 h and final conductivity was measured as ECf. The samples were autoclaved followed by cooling at 25 °C and used for measurement of total electrical conductivity (ECt). The following equation was used to calculation of electrolyte leakage:

EL (%): (EC_f-EC_i) / (EC_t-EC_i) × 100

Determination of proline

Proline content was measured by using literature methods (Bates et al.1973). Fresh leaf samples (0.5 g) were homogenized in 5 mL sulfosalicylic acid (3%) and centrifuged at 4000 rpm for 30 min. The supernatant was separated, and 1 mL each of glacial acetic acid and ninhydrin reagent were added to the supernatant (1 mL). The mixture was heated (100 °C) in oven for 1 h and then shifted to ice bath for reaction stoppage. Two milliliters toluene was added to the reaction product and kept at 25 °C until the appearance of 2 layers. Absorbance of the upper colored layer was recorded at 520 nm. Proline content was measured by following the formula:

Proline (μM/g FW) = [(μg proline/mL × mL toluene) / 115.5 μg μmol⁻¹]/[g sample/5].

Results

Total RNA from leaves of Arabidopsis thaliana (Col-0) was extracted and used for the synthesis of full-length complementary DNA (cDNA). The expected 1458 bp HsfA1d was produced when full-length complementary DNA (cDNA) of Arabidopsis thaliana was used as a template (Sup. 1a). Digestion of cloning vector, pUC57GW-CmRccdB with restriction enzymes, XhoI and NcoI resulted in 2804, 833, and 630 bp expected fragments (Sup. 1b). The insert (AtHsfA1d) was infused in linearized vector (purified pUC57GW) and successfully transformed into stellar competent cells. Digestion of entry clone with restriction enzymes, NcoI and BglII, resulted in four fragments (2828 bp, 807 bp, 391 bp, and 237 bp) while two fragments (3637 bp and 630 bp) were produced for empty vector with same set of enzymes (Sup. 1c). Sequence analysis with M13 primers revealed 100% homology of the entry clone with AtHsfA1d (Sup. 2). The newly synthesized entry clone has been shown in the Sup. 3.

Heat shock transcription factor, HsfA1d, was sub-cloned in plant expression vector (pGWB402Ω), followed by transformation to stellar competent cells (Sup. 4). The plasmid (pGWB402Ω-HsfA1d) extracted from transformed bacterial colonies produced three expected fragments (8665 bp, 2439 bp, and 391 bp) upon cleavage with NCOI, while digestion of pGWB402Ω (control) with the same restriction enzyme yielded two bands (9384 bp and 2294 bp) with the same enzyme (Sup. 5a). HsfA1d sub-cloned in plant expression vector has been shown in Sup. 5b. After restriction digestion-based confirmation, Agrobacterium GV3101 cells were successfully transformed with pGWB402Ω-HsfA1d (Sup. 6). Potato internodes infected with Agrobacterium harboring pGWB402Ω-HsfA1d exhibited growth on kanamycin containing media while no such growth was observed in wild type and ultimately died on selection media (Sup. 7a). Calli were produced, after 4 weeks, on MS media supplemented with ZR trans-isomer (0.8 mg L⁻¹) and 2,4-D (2.0 mg L⁻¹; Sup. 7b). In 3 weeks, the calli started regeneration on MS media supplemented with ZR trans-isomer (0.8 mg L⁻¹) and GA₃ (2.0 mg L⁻¹; Sup. 7c). The regenerated shoots produced in vitro tubers on MS media containing sucrose (60 mg L⁻¹), kinitin (2.5 mg L⁻¹) and gelrite (2.0 mg L⁻¹) in 1 month under complete darkness (Sup. 7d). Expected 1458 bp band was observed when transgenic DNA of putative transgenic plants was exposed to PCR with HsfA1d-specific primers while no such band was produced when DNA extracted from wild-type plants was used as template (Sup. 8).

Upon heat treatment, no obvious adverse effect on growth of transgenic plants was observed at phenotypic level while wild type showed stunted growth and turned yellowish (Fig. 1). The expression of target gene (HsfA1d) was witnessed in transgenic plants both at room temperature and thermal stress; however, the level of expression was more pronounced (5.5-fold) upon exposure to heat stress compared to room temperature (Fig. 2a). Transgenic plants exhibited 4- and 6-fold higher expression of HSP70 at room temperature and thermal stress respectively, compared to wild-type plants (Fig. 2b). Both transgenic and wild-type plants exhibited 2.2-fold increase in the expression HSP90 under heat stress compared to room temperature; however, no significant difference in their expression was recorded at room temperature (Fig. 2c). Heat stress caused significant reduction in chlorophyll a content of wild-type plants compared to room temperature while no such decline was recorded in transgenic plants (Fig. 3a). Significant decrease in chlorophyll b content of wild-type plants was observed upon exposure to thermal stress. On the other hand, no significant decrease in chlorophyll b content of transgenic plants was recorded under heat stress (Fig. 3b). Both wild-type and transgenic plants revealed no significant difference in their carotenoid pigments under thermal stress and room temperature (Fig. 3c). Wild-type and transgenic plants exhibited no significant difference in their EL at room temperature; however, highly significant and significant increase in EL of wild-type and transgenic plants, respectively, was recorded upon exposure to heat stress (Fig. 3d). Thermal stress induced significant increase in proline content of wild-type plants as compared to room temperature. Similarly, transgenic plants exhibited highly significant increase in their proline contents under thermal stress. However, both types of plants showed no significant difference in accumulated proline at room temperature (Fig. 3e).

Fig. 1.

Phenotypic effect of heat treatment (42°C) on wild type (WT) and transgenic potato (TP)

Fig. 2.

Effect of heat treatment on expression of target and downstream genes belong to wild type (WT) and transgenic plants (T.P). Data are shown as means of control and treatments from 3 independent replicates. Different letters indicate that the change is statistically significant as determined by 2 factorial ANOVA followed by LSD (p ≤ 0.05). Effect of heat treatment on expression of aHsfA1d, bHSP70, and cHSP90 of wild type (WT) and transgenic plants (TP)

Fig. 3.

Effect of heat treatment on different parameters of wild type (WT) and transgenic plants (T.P). Data are shown as means of control and treatments from 3 independent replicates. Different letters indicate that the change is statistically significant as determined by 2 factorial ANOVA followed by LSD (p ≤ 0.05). Effect of heat treatment on a chlorophyll ‘a’ content, b chlorophyll ‘b’ content, c carotenoids, d electrolyte leakage, and e proline content of wild type and transgenic plants

DiscussionGenetic engineering has provided a valuable tool in hands of molecular biologists to enhance crop tolerance against different stresses that ultimately leads to increase productivity. The knocking out of HsfA1d from Arabidopsis plants remarkably declines their thermotolerance (Hsiang-chin and Yee-yung, 2012). Similarly, HsfA1d overexpression has significantly enhanced the ability of model tobacco plant to counter the detrimental effects of heat stress (Shah et al.2017). The idea behind the current study was to investigate whether overexpression of HsfA1d enhance thermotolerance of the crop plant like potato. Agrobacterium-mediated transformation was used to generate transgenic potato plants overexpressing HsfA1d. Upon exposure to heat stress, wild-type plants showed reduced growth while transgenic plants exhibited more tolerance and hence no significant adverse effects on growth were recorded. The transgene (HsfA1d) incorporated in potato genome enabled the plantlets to remain green under thermal stress, while the wild-type plants could not cope with the harsh effects of heat stress and turned yellowish (Fig. 1).

The higher expression (5.5-fold) of target gene exhibited by transgenic plants under thermal stress (Fig. 2a) was in line with Qain et al. (2014). These results further endorsed the findings of Xue et al. (2010) that heat stress causes upregulation of transgene. The elevated expression of HSP70 recorded in transgenic plants at room temperature (4-fold) strengthened the reports of Higashi et al. (2013) that transgenic Arabidopsis overexpressing TsHsfA1d caused upregulation of many genes under normal growth temperature. Such results further ascertained the role of HsfA1d as transcription activator. The increase in expression of HSP70 under thermal stress (6-fold; Fig. 2b) was in agreement with Qain et al. (2014) that heat stress induces the expression of heat shock genes. These results showed that HsfA1d triggers the expression of downstream HSP70 more than HSP90 (Fig. 2c) at transcription level. The significant decrease in both chlorophyll a and b content exhibited by wild-type plants, under thermal stress, is indicative of heat-induced structural damage to the chloroplast. The decline in chlorophyll content, as recorded in Fig. 3a, b, has also previously been reported by Naz et al. (2018) in potato. The higher chlorophyll (a, b) retained by transgenic plants compared to wild type is in agreement with Xu et al. (2014). No effect of heat stress on carotenoids (Fig. 3c) content reflects their heat-stable nature and is in line with study of Mohamed and Aisha (2019). The magnitude of electrolyte leakage gives excellent clue about damage that happened to the plant cell membrane (Bajji et al.2002). Thermal stress has enhanced the electrolyte leakage of both types of plants; however, the extent of leakage was more pronounced in wild-type than transgenic plants as reflected in Fig. 3d. The lipid component of plasma membrane is particularly prone to thermal stress widening pores in cell membrane of wild-type plants associated with more leakage. In contrast, the cell membrane of transgenic plants was found to offer more protection against the damage caused by thermal stress leading to less electrolyte leakage (Shah et al.2017). The low level of EL shown by transgenic plants under thermal stress is also reported by Mohamed and Aisha (2019). Proline serves as excellent stress marker. The obvious increase in proline content of transgenic plants compared to wild type, in response to thermal stress (Fig. 3e), has strengthened the previous reports of Xue et al. (2010). It has been reported that enhanced accumulation of proline acts as protectant against thermal stress and reduce the damage to the cellular machinery (Gupta et al.2013).

Author’s contribution

Research work was conducted by Zamarud Shah under the supervision of Safdar Hussain Shah and Gul Shad Ali. Iqbal Munir and Asad Jan checked the manuscript time to time for improvement. Nisar Ahmed helped in figure setting. Raham Sher Khan and Arshad Iqbal contributed in revising the manuscript.

Funding information

Financial support for the current study was extended by two Pakistani organizations (HEC, Islamabad and UST, Bannu). Research facilities were jointly provided by MREC, University of Florida, USA and IBGE, University of Agriculture Peshawar Pakistan.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.