The functional analysis of a wheat group 3 late embryogenesis abundant protein in Escherichia coli and Arabidopsis under abiotic stresses

ABSTRACT

Late embryogenesis abundant (LEA) proteins are highly hydrophilic and thermostable proteins that could be induced by abiotic stresses in plants. Previously, we have isolated a group 3 LEA gene WZY3-1 (GenBank: KX090360.1) in wheat. In this study, the recombinant plasmid with WZY3-1 was transformed into Escherichia coli BL21 for protein expression. Furthermore, we transformed WZY3-1 into Arabidopsis. Overexpression of WZY3-1 in E.coli enhanced their tolerance to mannitol and NaCl. WZY3-1 protein could protect lactate dehydrogenase (LDH) under freeze and heat stress. Overexpression of WZY3-1 showed that WZY3-1 could help to improve the drought tolerance of transgenic Arabidopsis. In summary, our works show that WZY3-1 plays an important role in abiotic stress resistance in prokaryotic and eukaryotic organisms.

KEYWORDS:

• LEA protein
• drought stress
• NaCl stress
• transgenic Arabidopsis
• wheat

Introduction

Late embryogenesis abundant (LEA) proteins, originally found in cotton seeds, belong to a large group protein family that maintain the water content in plant cells under abiotic stresses.¹ During abiotic stresses (drought, cold, high salinity, etc.), the accumulation of LEA proteins increased dramatically in various organs of the plant and some LEA proteins accumulation position varied after stresses.² An Arabidopsis LEA protein ERD14 mainly expressed in leaf, root and stem under normal condition. However, under cold stress, the accumulation of ERD14 was detected throughout the entire plant.³ Most of LEA proteins are hydrophilic and thermostable. They possess high ratios of charged amino acids such as Lys and Gly and lack of uncharged amino acid like Cys and Tyr. Although the protective mechanisms of LEA proteins are unclear, many studies have indicated that LEA protein can inhibit aggregation of proteins/enzymes, and bind to excessive metal ions and lipids under low temperature and desiccation^4–8

LEA proteins can be classified into seven protein subfamilies, among which the group 3 of LEA protein are identified by an 11-mer amino acid motif (TAQAAKEKAGE)⁹ and the conserved 11-mer amino acids play an important role in LEA 3 proteins acquiring α-helical conformations during dehydration.^10-13 Both in silico predictions and spectroscopic experiments demonstrate that LEA 3 proteins are mainly intrinsically disordered proteins (IDPs). Some study on LEA 3 proteins also revealed that a typical IDP AcLEA could protect E.coli against desiccation, oxidant, and osmotic stresses through clear conformational variation detected by circular dichroism (CD), Fourier transform infrared spectrometer (FTIR) and nuclear magnetic resonance (NMR) (Saucedo et al., 2017). The model peptide of group 3 LEA protein G3LEA from the larvae of P. vanderplanki is involved in the structural transition from random coils to α-helix in drought stress.¹⁰ Study on TdLEA3 showed that drying, trifluoroethanol (TFE) and glycerol were capable of inducing α-helical structure from disordered state of TdLEA3. Moreover, TdLEA3 was able to prevent the inactivation of lactate dehydrogenase (LDH) under heat, dehydration-rehydration and freeze-thaw treatments via reducing the aggregate formation of LDH.¹⁴ ZmLEA3 could participate in preventing aggregation of α-casein, LDH via disordered-to-order conformations in vitro, in addition, ZmLEA3 could improve low-temperature tolerance in transgenic yeast, E.coli, and overexpression plants.⁶ Overexpression of a wheat LEA gene in Phellodendron amurense indicated that TaLEA3 could improve the drought resistance by inducing rapid stomatal clousure in transgnic lines, and further experiments indicating that TaLEA3 overexpression could positively regulate nitric oxide (NO) biosynthesis and accumulation in the guard cells.¹⁵ Similar to TaLEA3, a LEA gene named AtLEA3 from Arabidopsis also enhanced the drought tolerance in overexpression lines, and seed size, membrane stability and seed weight were also improved in overexpression lines.¹⁶

Although previous works have revealed part functions of LEA 3 proteins, the roles of LEA 3 in plant abiotic stress tolerance are still ambiguous. In this work, overexpression of WZY3-1 in E.coli and Arabidopsis resulted in improved tolerance to drought and salt stress.

Materials and methods

Expression of WZY3-1 in E.coli

The cDNA of WZY3-1 was kept in our lab. WZY3-1 was inserted into the PET28a vector and then transformed into E.coil BL21(DE3) in order to obtain the recombinant proteins of WZY3-1. The recombinant protein with His-tag at N-terminus was purified through Ni²⁺ Heparin-Sepharose affinity chromatography and then identified by Western blot. His antibody was selected for the Western bolt.

LDH protection assay

10 μg/mL LDH (Sigma, USA) from rabbit muscle was mixed with 10 mM sodium phosphate buffer (pH 7.4) and WZY3-1 protein. In the low-temperature assay, the mixture was frozen for 1 min in liquid nitrogen and thawed for 10 min in a water bath at 25°C. In the high-temperature assay, the mixture was heated for 30 min in a water bath at 45°C. The reaction mixture contained 10 mM pyruvic acid and 2 mM nicotinamide adenine dinucleotide (NADH). LDH activity was measured as the rate of decrease in absorbance at 340 nm for 5 min using a microplate reader (SpectraMax M2, Molecular Devices, USA) due to the oxidation of NADH. The rate determined for the untreated samples was considered as 100% in our experiments.

Abiotic stresses tolerance assays of E.coli transformants

The recombinant E.coil was cultured in lysogeny broth (LB) medium containing 0.5 mM isopropyl β-D-thiogalactoside (IPTG) for 6 h, then 400 μL E.coil solution was added to 15 ml LB contains 500 mM NaCl and 1 M mannitol, respectively. At each time point, 3 ml of culture was used to measure the absorbance at 600 nm. For temperature treatment, the culture was cooled in −20°C for 2h or heated in 50°C for 1 h.

Plant materials and growth condition

The Columbia Arabidopsis was used in this study. The Arabidopsis seeds were grown in a commercial soil mix under 200 μE m⁻²s⁻¹ light at 20/23°C (night/day) temperature with a 16-h photoperiod.

Arabidopsis transformation and abiotic stress tolerance analysis

WZY3-1 was cloned into PBI121 and driven by CaMV 35S promoter. The construct was then transferred into Agrobacterium GV3101. The Arabidopsis plants were transformed by the floral dip method¹⁷ and homozygous T3 seeds were used.

For osmotic stress assay, wild-type and transgenic Arabidopsis were cultivated into 1/2 Murashige and Skoog (MS) medium for 10 d, then transferred them into the new 1/2 MS vertical medium that contained 0 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM mannitol, respectively. For drought stress treatment, Arabidopsis ecotype Columbia plants and transgenic Arabidopsis were cultivated in the soil for 4 weeks and then stop to the water supply.

Measurements of malondialdehyde (MDA), proline, chlorophyll and water content

Rosette leaves of Arabidopsis were gathered to measure the physiological parameters. MDA contents and proline contents were evaluated as described previously.¹⁸ Chlorophyll contents were measured as described previously.¹⁹ For water content assay, the samples were dried at 98°C to 100°C until the constant dry weight was achieved, generally expressed on a percentage fresh weight basis.

Quantitative RT-PCR analysis

In order to detect the expression pattern of WZY3-1, CPK3, CPK6, GHR1, HT1, OST, and SLAC1, total RNA was isolated from Arabidopsis with or without treatment (trizol). cDNA was synthesized from RNA using TransScript One-Step gDNA Removal and cDNA Synthesis Supermix (TransGen Biotech, Beijing) according to its manual. QRT-PCR was implemented using BIO-RAD CFX96 and TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing). Each sample was measured three biological replicates.

Stomatal aperture

Three leaves of wild-type and WZY3-1 transgenic Arabidopsis were cut and the lower surface was immediately stuck to a microspore slide with a medical adhesive. After 1 min, the leaf was peeled away under distilled water. The lower epidermis stuck to the glass was visualized under the microscope and stomatal images were recorded. Measurements of stomatal aperture were performed using the imaging software Image J, version 6.

Statistical analysis

All of the experiments in this study were repeated three times in independent experiments, and the data shown are the mean ± SD. In this research, statistical analyzes were performed using the statistical tools (Student’s t-test) of Microsoft Excel software.

Results

Purification and acquisition of WZY3-1 protein

Prokaryotic expression method was used in our assay to obtain the WZY3-1 protein. The protein was purified rely on His-tag of its terminal (Figure1a). To further validate the recombinant protein is WZY3-1, we carried out Western blot assay and the results demonstrate that we obtained the WZY3-1 protein and the molecular weight confirmed to our projection (Figure1b).

Figure 1. Analysis of induced expression products by SDS-PAGE (a) and Western Blot (b).

(a) M: Protein Marker; 1: The supernatant obtained after heat from E. coli cultures producing pET28a with IPTG; 2: The supernatant obtained after heat from E. coli cultures producing pET28a without IPTG; 3: The supernatant obtained after cell disruption from E. coli cultures producing WZY3-1; 4: The supernatant obtained after cell disruption and heat; 5: WZY3-1 protein purified with Ni2+ agarose gel affinity column. (b) M: Protein Marker; 1–3: protein purified with Ni2+ agarose gel affinity column.

WZY3-1 protected ldh activity under freezing and thermal stress conditions

Since LDH loses its activity during freezing and thermal conditions and a number of LEA proteins can protect LDH protein. We selected this enzyme to evaluate the protective effect of WZY3-1. LDH activity before treatment was regarded as 100%. After freezing treatment, the activity of LDH was 97% in the presence of WZY3-1. In contrast, the activity of LDH was only 27% in sodium phosphate buffer. For thermal treatment, similarly, the activity of LDH was 26% in sodium phosphate buffer but in the presence of WZY3-1, the activity of LDH higher than 80% (Figure 2).

Figure 2. Protection of WZY3-1 protein on LDH activity under freezing and heat treatment. The of LDH activities were measured under freezing (f1, f2, f3) and heat (ht1, ht2, ht3) treatment with three cycles. Asterisks indicate statistically significant differences (*P < .05, **P < .01; Student’s t-test).

In summary, obviously, WZY3-1 can protect the activity of proteins from damage caused by freezing and thermal stresses.

Overexpression of WZY3-1 enhanced the viability of E.coli under abiotic stresses conditions

To explore whether WZY3-1 protein could enhance abiotic stress tolerance in vivo, the growth of the recombinant E.coil was examined to represent the function of WZY3-1 in abiotic stress. Under optimal conditions, there was no obvious growth difference the control and transformed lines. However, in the presence of 400 mM NaCl or 400 mM mannitol, the growth condition of the transformed line was superior to the control (Figure 3a–c). Similarly, in freezing or thermal stresses, the transformed line showed higher viability than the control after culture for 24h (Figure 3d,e).

Figure 3. The growth curve of E. coli strains producing WZY3-1 protein and pET28a under normal conditions (a) or mannitol (b) and NaCl (c) treatments, with the growth state of E. coli strains producing WZY3-1 after −20°C (d) and 50°C (d) treatment.

Overexpression of WZY3-1 enhanced transgenic arabidopsis tolerance to abiotic stresses

To further elucidate the function of the WZY3-1 protein in plant tolerance to abiotic stresses, independent transgenic lines were obtained by Kanamycin-resistance selection and confirmed by genomic PCR. The semi-quantitive RT-PCR assay was used to detect the expression levels of WZY3-1 in Arabidopsis (Figure 4a). Three independent WZY3-1 transgenic lines (T3-1, T3-5, T3-7), with higher transgenic expressions, were detected for their tolerance under abiotic stresses. In mannitol treatment assay, when the concentration of mannitol exceed 150 mM, the root length of the transgenic plants longer than WT plants until the concentration of mannitol more than 300 mM (Figure 4b,c). After drought stress treatment, the transgenic lines exhibited greener leaves than WT plants (Figure 4d). These results indicate that overexpression of WZY3-1 increases plants tolerance to osmotic and drought stress. To further confirm the tolerance of transgenic lines in drought stress, physiological and biochemical indexes of transgenic plants was measured, the results show that the water content and chlorophyll content of transgenic plants was higher than WT plant after drought treatment, it is noteworthy that the proline content of transgenic lines showed a sharp rise. On the contrary, the MDA contents of transgenic lines were lower than WT plants (Figure 5).

Figure 4. The screening of transgenic Arabidopsis. (a) The semi-quantitative test of wildtype (WT) and transgenic Arabisopsis (line 3–1, 3–5 and 3–7). (b) The growth state of WT and transgenic Arabisopsis under mannitol treatment. (c) The root length of WT and transgenic Arabidopsis under mannitol treatment. (d) The growth state of WT and transgenic Arabidopsis under drought stress for 0, 6, 7, 8 and 10 days. Asterisks indicate statistically significant differences (*P < .05, **P < .01; Student’s t-test).

Figure 5. Measurement of physiological and biochemical indexes of transgenic and wild type plants treated with drought. (a) The water content of WT and transgenic Arabidopsis. (b) The total chlorphyll content of WT and transgenic Arabidopsis. (c) The proline content of WT and transgenic Arabidopsis. (d) The MDA content of WT and transgenic Arabidopsis. Asterisks indicate statistically significant differences (*P < .05, **P < .01; Student’s t-test).

In addition, we also measured the stomatal aperture of WT and transgenic Arabidopsis under normal and ABA/Dehydration treatment (Figure 6). The results showed a decreased aperture of stomata in both WT and transgenic plants under 1h ABA and 40 min dehydration treatment. However, the stomata apertures in transgenic plants are lower than WT (Mock) lines. To further elucidate the relationship between WZY3-1 and stomata closure, we implemented qPCR of seven stomatal movement-related genes, including calcium-dependent protein kinase 3 (CPK3), calcium-dependent protein kinase 6 (CPK6), Guard Cell Hydrogen Peroxide-resistance 1(GHR1), HIGH LEAF TEMPERATURE1 (HT1), OPEN STOMATA (OST1) and SLOW ANION CHANNEL1 (SLAC1) (Figure 7). The results of qPCR demonstrated the expression of GHR1, HT1 and OST1 were significantly up-regulated in transgenic plants under drought conditions. Taken together, these results demonstrated that WZY3-1 was involved in plant tolerance to abiotic stresses.

Figure 6. Stomata aperture of wild type and transgenic Arabidopsis. (a) The stomaltal aperture measurements of WT and transgenic lines under normal (Mock), ABA and dehydration treatment. (b) Observation of stomata of WT and transgenic line.

Figure 7. The expression of stomata closure related genes. Asterisks indicate statistically significant differences (*P < .05, **P < .01; Student’s t-test).

Discussion

At present, a series of studies have uncovered that LEA3 proteins could protect the plant from environmental stresses by their unique protein structure and intrinsically disordered property.^4,6,7,20,21 LEA proteins are highly hydrophilic, and are speculated to protect other proteins/enzymes from aggregation or desiccation.²² LDH as a reporter enzyme have been widely used to test the protection activity of different group LEA proteins, and it is hypothesized that this protection activity is implemented via chaperone molecules of LEA protein.^14,22,23 In our study, the result showed that WZY3-1 could protect the LDH activity from damage caused by freezing and heat cycles (Figure 2). Previous study indicated that LEA protein could function via formation of dimers that forming a large molecular shield around biomolecules. The result indicated WZY3-1 may protect LDH activity by stabilizing enzyme under environmental stress.

Overexpression of LEA genes in many plants has successfully indicated their enhancement of abiotic stress tolerance.^6,7 Overexpression of a barley LEA gene HVA1 in transgenic rice improved the tolerance to water deficit and salt stress.²⁴ Further study also showed overexpression of HVA1 could enhance the growth of rice root.²⁵ Recent analysis of transgenic study confirmed that LEA3 could alter lipid accumulation via enhancing photosynthetic efficiency and reducing ROS content.¹⁶

In this study, the transgenic Arabidopsis showed longer root length under osmotic stress, indicating that WZY3-1 could enhance transgenic Arabidopsis tolerance to osmotic stress. Furthermore, the transgenic Arabidopsis also exhibited higher water content, proline total chlorophyll, and lower MDA content. According to those results, we could speculate that WZY3-1 could enhance transgenic Arabidopsis tolerance to drought stress.

Group 3 LEA proteins are not only widely distributed in the eukaryotic organisms, but also existed in prokaryotes such as Deinovoccus radiodurans²⁶ and Acidovorax avenae.²⁷ MeLEA3 from Manihot esculenta is charaterised to confer a protective function against heat and salt stress in bacterial cells.²⁸ To investigate whether WZY3-1 could protect prokaryotic organisms, E.coli BL21 was chosen for further study. The expression of WZY3-1 in E.coli significantly increased its tolerance to osmotic, salt, heat and cold stresses. The results implying that some common protective mechanisms in both prokaryotes and eukaryotes under abiotic stress. In addition, we also observed stomatal closure of Arabidopsis under ABA treatment. Our results showed the stomatal apertures of transgenic Arabidopsis were significantly reduced to wild-type.

Stomatal aperture is largely regulated by cell turgor, which is controlled by solutes and water in guard cells. The stomatal closure is caused by efflux of anion through anion channels in guard cell plasma membrane, the plasma membrane are then depolarized and thr outward cation channels are activated, which results in efflux of solutes and decreased turgor in guard cells.²⁹ The protein kinase HT1 has a central role in CO2-induced staomatal regulation.³⁰ Other than HT1, several guard cell regulators have also been characterized. The disruption of SLAC1 expression could impaired the stomatal closure induced by ozone, CO2, ABA and calcium, which implied that the SLAC protein family is essential for the plasma membrane of S-type anion channel function in guard cells.^31,32 SLAC1 could be phosphorylated by OST1, which is a serine/threonine protein kinase that positively regulate ABA-mediated stomatal responses.³³ Smilar to OST1, a gene named GHR1, which encodes a receptor-like kinase localized on the plasma membrane in Arabidopsis, could also interact and activate S-type anion channel SLOW ANION CHANNEL-ASSOCIATED1.³⁴ Further study also uncovered that SLAC1 HOMOLOG3 (SLAH3) also contributes to S-type anion channel and essential for nitrate transport in the roots.^35,36 Recent study discovered that SLAC1/SLAH3 participated in signaling pathways for stomatal closure and opening in Arabidpopsis.³⁷ CPKs are widely distributed in the plant, and CPKs appear to play a dual role in abiotic stress response. Genetic study have uncovered that AtCPK3 and AtCPK6 mediate ABA responses in guard cells, and they participate in calcium activation in slow-type anion channels.³⁸ In addition, AtCPK3 is found to contribute to salt stress by phosphorylating the vacuolar two-pore K+ channel (TPK1).³⁹ In our study, the qRT-PCR of stomatal movement related genes also implied that overexpression of WZY3-1 could up-regulate GHR1, HT1 and OST1 (Figure 7), which showed a positive role of LEA proteins in enhancing the closure of stomata under abiotic conditions and ABA treatment, which is coherent to CaLEA1 and TaLEA3.^15,40 However, the WZY3-1 could regulate the stomatal movement without the significant expression of CPK3, CPK6 and SLAC1, indicating a complex regulation of LEA gene and ABA-mediated stomatal movement that is essential to explore in futher study.

In summary, our works show that WZY3-1 plays an important role in abiotic stress resistance in E.coli and Arabidopsis, but the molecular mechanism to elucidate how WZY3-1 participated in abiotic stress responses are still ambiguous, thus, further experiments are necessary to explore this question.

Authors’ contributions

LZ and ZY designed the study, wrote part of the manuscript, and finalized the figures. XW and YT carried out most of the experiments and data analysis and wrote part of the manuscript. ZY, XW, YT and DZ performed experiments and took care of plants. All authors have read and approved the manuscript.

Disclosure of potential conflicts of interest

The authors declare no conflict of interest.

Additional information

Funding

This work was financially supported by National Natural Science Foundation of China [31671608], and the Fundamental Research Funds for the Central Universities [Z109021567].