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Investigating the role of Bowman-Birk serine protease inhibitor in Arabidopsis plants under drought stress
Plant Physiology and Biochemistry 149 (2020) 286–293
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Investigating the role of Bowman-Birk serine protease inhibitor in Arabidopsis plants under drought stress
T
M.B. Malefoa,b, E.O. Mathibelaa,b, B.G. Cramptona,b, M.E. Makgopaa,b,∗ a b
Department of Plant and Soil Sciences, Private Bag X20, Hatfield, Pretoria, 0001, South Africa Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, Hatfield, 0028, South Africa
A R T I C LE I N FO
A B S T R A C T
Keywords: Bowman-birk inhibitor Serine protease inhibitor Drought tolerance Arabidopsis Antioxidant enzymes
Serine protease inhibitors (SPIs) play an important role in cell survival, development and host defense. In plants, serine protease inhibitors such as the Kunitz-type inhibitor (KTI) and the Bowman-Birk inhibitor (BBI) have been shown to be induced in response to abiotic stress such as salinity and drought resulting in tolerance to these stresses. In this study, Arabidopsis thaliana (T3) plants overexpressing the BBI gene from maize were generated and subjected to drought stress in order to study the role of BBI protease inhibitor in drought tolerance. Drought treatment of four-week-old Arabidopsis plants was performed by withholding water from plants for nine days and harvested plant material was used for physiological and biochemical analysis. The transgenic lines exhibited normal growth after nine days of drought as compared to the wild-type. The results also showed a higher leaf relative water content (RWC) in transgenic lines when compared to the wild-type (WT), with line 2 having the highest RWC of 72% and the WT having the lowest RWC of 32%. Trypsin-inhibitor activity indicated that the total protein of the positive transgenic plants had stronger protease inhibitory activity than the wild-type. Transgenic lines overexpressing BBI also showed reduced lipid peroxidation (MDA content) as well as enhanced activity of antioxidants glutathione-s-transferase (GST) and ascorbate peroxidase (APX). These results suggest that BBI protease inhibitor leads to drought tolerance associated with reduction in drought-induced oxidative stress.
1. Introduction Plants are often exposed to abiotic stresses such as drought, salinity, cold, heat and extreme light which pose a threat to plant production and growth. These abiotic stresses are responsible for crop losses worldwide, resulting in yield reductions of over 50% (Ahmad et al., 2016). Availability of water is one of the key factors that contribute to the reduction of crop production. Drought is thus one of the mostly studied abiotic stresses as it severely affects the growth and development of plants. The effect of drought in plants is observed at the cellular level, where it disrupts cellular functions (Bray, 2001), and it also affects the photosynthetic parts of the plant, which results in reduced photosynthetic rates, destroyed chloroplast structures and restrictions to electron transport and enzyme activity (Zhou et al., 2015). The study of variations in gene expression during drought is commonly used to resolve the molecular basis of drought stress leading to either susceptibility or tolerance to drought. For many years
Arabidopsis has been used as a model plant, linking cellular responses and gene expression data. Together with soybean and tobacco, Arabidopsis has been used for the improvement of water use efficiency in crops and the regulatory genes underlying the efficiency have been studied in these plants (Zhou et al., 2015). Studies have shown that post-translational regulation can result in poor correlations between transcripts and their proteins in the transcriptomic analysis of samples under drought conditions and thus, proteomics can be used as a powerful tool in exploring the response mechanisms of plants to various stresses (Ali and Komatsu, 2006). Protein breakdown has been identified as one of the key adaptations of plants to abiotic stress (Budič et al., 2016). In plants, a group of proteolytic enzymes called proteases are responsible for protein breakdown. Proteases hydrolyze specific peptide bonds in proteins, and they are classified based on the type of active site residue they possess i.e. cysteine, metallo-, aspartic, glutamic, asparagine, and serine proteases (Barrett, 1986). These proteolytic enzymes are responsible for
Abbreviations: RWC, relative water content; BBI, Bowman-Birk inhibitor; SPI, serine protease inhibitor; PI, protease inhibitor; GST, glutathione-S transferase; APX, ascorbate peroxidase; H2O2, hydrogen peroxide; MDA, malondialdehyde ∗ Corresponding author. Department of Plant and Soil Sciences, Private Bag X20, Hatfield, Pretoria, 0001, South Africa. E-mail address: [email protected] (M.E. Makgopa). https://doi.org/10.1016/j.plaphy.2020.02.007 Received 19 October 2019; Received in revised form 8 February 2020; Accepted 9 February 2020 Available online 15 February 2020 0981-9428/ © 2020 Elsevier Masson SAS. All rights reserved.
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resistance to the herbicide phosphinothricin and its derivatives. Agrobacterium-mediated transformation was performed according to the floral dip method (Clough and Bent, 1998). Wild-type A. thaliana (ecotype Col-0) plants were grown in controlled environment chambers, at day/night temperatures of 25°C/20°C and an irradiance of 400 μmol m−2s−1 with a 16h photoperiod. Primary inflorescence buds were clipped to allow the formation of secondary inflorescence buds and increase the transformation process by obtaining more flower buds per plant. Plants were grown for 8 weeks before floral dip transformation with Agrobacterium tumefaciens strain GV3101 carrying the plasmid pTF101.1-BBI (Clough and Bent, 1998). Secondary inflorescences were submerged into the A. tumefaciens cell suspension for 10 s and then allowed to grow in the controlled environment chambers until seed maturity.
cellular housekeeping and stress response by degrading misfolded and potentially harmful proteins, to provide amino acids for the production of new proteins (Gur et al., 2011). Proteases play a major role in protein breakdown during leaf senescence under biotic and abiotic stresses. Proteolytic activity also increases during drought stress as a result of hydrogen peroxide production leading to the enhancement of leaf senescence and drought stress sensitivity (Dhanushkodi et al., 2018). Studies by Chen et al. (2013), reported enhanced drought sensitivity in transgenic lines overexpressing the sweet potato cysteine protease gene, SPCP3. Evidence of papain-like cysteine proteases that lead to oxidative damage and programmed cell death (PCD) have been previously reported, indicating the involvement of proteases in abiotic stress susceptibility (Lampl et al., 2013). Serine proteases are the most prominent of the seven families of proteases in plants. Serine proteases catalyze the hydrolysis of specific peptide bonds in their substrate, which contain a serine amino acid residue in the active site. This family of proteases is involved in many physiological processes which includes the hypersensitive response, signal transduction, senescence and protein degradation (Asif-Ullah et al., 2006). A recent study by Fan et al. (2016) also suggests that serine protease expression could be involved in defense response against biotic and abiotic stresses. Despite being important for cellular functions, proteases can be very damaging when overexpressed, leading to degradation of many proteins in response to environmental factors and as a result, they are strictly regulated. Cell damage by proteases leads to the expression of protease inhibitors (PI) which are used to control the unwanted proteolysis by proteases (Yang and Yeh, 2005). Protease inhibitors are plant proteins which function by regulating proteolytic activity of their target proteases, resulting in the formation of a stable protease inhibitor complex (Meenu and Murugan, 2016). Protease inhibitors have also been shown to play an important role in abiotic stress tolerance. In Arabidopsis thaliana, cysteine protease inhibitors were noted to increase salt, drought, oxidation and cold tolerance (Zhang, 2008). Serine protease inhibitors are well-known defense components in plants, and they are responsible for inhibiting trypsin and chymotrypsin. Plant inhibitors such as the Kunitz-type inhibitor (KTI) and Bowman-Birk inhibitor (BBI) genes, encoding serine protease inhibitors have been shown to play a role in plant defense against insects and pathogens (Dramé et al., 2013). The induction of BBIs has been observed during abiotic stresses such as salinity and metal toxicity. In maize, BBI gene transcript expression was previously found to be induced by high carbon dioxide concentration (Prins et al., 2011). However, the exact role of this protease inhibitor remains unknown. Although drought is one of the well-studied stresses, the involvement of protease inhibitors in enhanced tolerance has not been thoroughly studied. In this study, the BBI gene was overexpressed in Arabidopsis thaliana and its effect on improving drought tolerance in transgenic A. thaliana was evaluated.
2.2. Selection of transgenic lines 2.2.1. Plant growth and maintenance Transgenic T0 Arabidopsis seeds were sterilized in 70% ethanol for 5 min and in 10% bleach containing 0.1% Triton X for 15 min then washed with sterile water three times and re-suspended in 0.1% agar and placed on MS medium containing 50 mg/l Basta®. The plates were incubated at 4°C for 2 days in the dark and grown in constant light at 25°C for 7 days. Seedlings were transferred to moistened peat pellets (Jiffy product) and maintained until they were four weeks old. The plants were grown to maturity in a controlled growth room, at full day conditions of 16 hr light/8 hr day photoperiod at a temperature of 20–23°C and humidity of 45% RH. The following studies were performed on three independent transformed homozygous lines (Line 1, Line 2, and Line 3) and the wild-type (WT). 2.2.2. PCR amplification of the maize BBI gene Amplification of the BBI was performed using the Bio-Rad S1000™ Thermal Cycler (Bio-Rad Laboratories, California). A PCR product of 350 bp was amplified using maize cDNA specific BBI primers; forward primer 5′-GCC AGG ACA GGA GAA ACA AA-3′ and reverse primer 5′CAT GCC GTA CGT CAG AAG AA-3’. PCR was performed at 30 cycles using a 50 ng gDNA template per reaction in a PCR tube with DreamTaq PCR Master Mix (2X) (ThermoFisher Scientific, Johannesburg) in 25 μl reaction volume containing 0.5 μM forward and 0.5 μM reverse primers. PCR conditions were 1 cycle denaturation step at 94°C for 3 min; 30 cycles denaturation/annealing/elongation at 94°C for 1 min/60°C for 1 min/72°C for 1 min followed by 1 cycle final extension at 72°C for 10 min. The PCR products were verified by agarose gel electrophoresis analysis and then used for sequencing to confirm the BBI sequence (Sanger et al., 1977). 2.2.3. RNA extraction and RT-qPCR analysis Total RNA was extracted from the fully expanded leaves by ZymoBIOMICS™ RNA Mini Kit (Inqaba Technical industries, Pretoria) following the manufacturer's instructions. The RNA concentration was calculated from the optical density (OD) of the purified RNA at 260 nm wavelength using the Nano-Drop™ spectrophotometer (ThermoScientific, Waltham, Massachusetts) and 1 μg of RNA was transcribed to cDNA using the ReverseAid first strand cDNA synthesis kit transcriptase (ThermoFisher Scientific, Johannesburg). Real-time PCR was performed for the BBI gene using the gene-specific primers: 5′CAT CCG GAG TGC ATG AAC T-3′ and 5′-GTT TGT GAG AAC GTC GTC AC-3’. RT-qPCR assays were performed using a CFX96 Touch™ RealTime PCR Detection System in a 96-well Clear Multiplate PCR Plates (Bio-Rad, California). Each 10 μl reaction comprised 1 μl cDNA template, 5 μL KAPA SYBR® FAST qPCR Master Mix (Lasec SA (Pty) Ltd), Cape Town, 0.2 μM of each primer and 4 μL of sterile distilled water. For the non-template control, cDNA was replaced with sterile distilled water. The reactions were subjected to an initial denaturation step at 95°C during 10 min followed by 45 cycles at 95°C for 10 s, 60°C for 30 s
2. Materials and methods 2.1. Constructs and A. thaliana transformation The BBI gene (Accession number EF406276.1) was amplified from maize (Zea may L.) cDNA with gene-specific primers containing restriction enzyme sites BamHI and HindIII; BBI-HindIII forward primer 5′CCCG AAG CTT GCC AGG ACA GGA GAA ACA AA-3′ and BBI-BamHI reverse primer 5′-TCCT GGA TCC CAT GCC GTA CGT CAG AAG AA-3’. The amplicon was purified and cloned into pLBR 19 plasmid. The expression cassette of plasmid pLBR-BBI was sub-cloned into the SacI HF™ and XbaI restriction sites of plasmid pTF101.1 containing a spectinomycin resistant marker gene (aaadA) for bacterial selection. The plant expression cassette used contained the cauliflower mosaic virus (CaMV) double 35S promoter (2 × 35S) and the phosphine acetyltransferase gene (bar gene) from Streptomyces hygroscopicus which conferred 287
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2.7. Determination of lipid peroxidation
and 72°C for 15 s. The transcript level of BBI gene was estimated relative to four Arabidopsis reference genes; EFα: 5′-CAC CAC TGG AGG TTT TGA GG-3′ and 5′-TGG AGT ATT TGG GGG TGG T-3’ (GenBank accession At5g60390), SAND 5′-GTT GGG TCA CAC CAG ATT TTG and 5′-GCT CCT TGC AAG AAC ACT TCA-3, (GenBank accession At2g28390), PDF2 5′-TCA TTC CGA TAG TCG ACC AAG and 5′-TTG ATT TGC GAA ATA CCG AAC-3’ (GenBank accession At1g13320) and FBOX 5′-GGC TGA GAG GTT CGA GTG TT-3′ and 5′-GGC TGT TGC ATG ACT GAA GA-3’ (GenBank accession At5g15710). Relative gene expression levels were calculated using the 2−ΔΔCT method with three independent biological replicates (Livak and Schmittgen, 2001).
Lipid peroxidation was determined as previously described by Zhang et al. (2007). Whole Arabidopsis rosettes (100–700 mg) were ground in liquid nitrogen and placed into microfuge tube. Five volumes of 6% (w/v) trichloroacetic acid (TCA) (Merck Millipore, Germiston) was added to each microfuge tubes. The samples were homogenized using a vortex and then centrifuged at 10 000 g for 10 min to pellet the plant material. Approximately 200 μl of supernatant was transferred to a new microfuge tube followed by the addition of 300 μl of 0.5% (w/v) thiobarbituric acid (TBA) (Sigma, Aldrich, Johannesburg). The samples were mixed by brief vortexing. A small hole was punched on the microfuge lids to prevent the lids from opening under high temperature incubation. The sample tubes were placed in a heating block and were allowed to incubate at 90°C for 20 min. Following the 20 min incubation, the samples were incubated on ice for 10 min. Once the incubation was complete, the samples were centrifuged at 10 000 g for 5 min to pellet plant material. All extracted samples were then loaded in triplicate on a 96-well micro-titer plate. The plate was then read at 532 nm and 600 nm on a PowerWave™ microplate spectrophotometer (BioTek® instruments, INC, Winooski). The absorbance at 600 nm was subtracted from the absorbance at 532 nm to correct for the non-specific turbidity. Lipid peroxidation was determined by measuring activity of malondialdehyde (MDA) using spectrophotometry applying the extinction coefficient 155 Mm cm−1. Percentage increase was calculated by the equation:
2.3. Drought tolerance analysis of transgenic plants Seedlings (T3) of wild-type and transgenic plants (approximately 30 of each line) were germinated on moistened peat pellets (Jiffy product) and grown under short-day conditions (8 h light/16 h dark) at 22–24°C with normal watering for four weeks. The four-week-old plants were subjected to drought stress by withholding water for nine days thereafter, photos were taken, and plants were harvested for RNA and protein extractions. The whole rosette was harvested and then weighed for protein measurements. 2.4. Determination of relative water content For the quantification of relative water content, the whole rosette was collected from both well-watered and drought-treated plants and immediately weighed for fresh weight. The leaf material was then placed at 80°C overnight then weighed the samples for dry weight measurements. The relative water content was calculated by the equation (Quain et al., 2014):
RWC % =
fresh weight − dry weight X 100 fresh weight
%change =
Xwell − watered − Xdrought Xwell − watered
X 100
(2)
2.8. Hydrogen peroxide (H2O2) content A modified method of Velikova et al. (2000) was followed to determine the H2O2 content in the plant material. Whole Arabidopsis rosettes (100–700 mg) were ground in liquid nitrogen and inserted in a microfuge tube. Five volumes of 6% (w/v) trichloroacetic acid (TCA) (Merck Millipore, Germiston) was added to the microfuge tubes. The samples were homogenized using a vortex and then centrifuged at 10 000 g for 10 min to pellet the plant material. Approximately 200 μl of supernatant was transferred to a new microfuge tube followed by the addition of 300 μl of 0.5% (w/v) thiobarbituric acid (TBA) (Sigma, Aldrich, Johannesburg). The samples were mixed by brief vortexing. Standards from a 30% H2O2 stock (Sigma Aldrich, Johannesburg) ranging from 10, 20, 30, 40, 50, 60, 70, 80, and 100 μM were prepared by diluting an appropriate volume of H2O2 in deionised water and used to measure H2O2 in extracted samples. A volume of 50 μl extracted sample and standards were then loaded in triplicate on a 96-well microtiter plate. To the samples and standards, 1.25 mM dipotassium hydrogen phosphate (K2HPO4) and 250 mM potassium iodide (KI) were added. Once the plate was prepared and the solutions properly mixed, it was incubated for 20 min at room temperature. The samples were then read at 390 nm using a PowerWave™ microplate spectrophotometer (BioTek® instruments, INC, Winooski).
(1)
2.5. Protein extraction and yield determination Total protein extracts were obtained from leaf rosette of transgenic and wild-type Arabidopsis plants. The leaves (100 mg) were ground into a powder and dissolved in 50 mM Tris-HCl, pH 8 (Sigma, Aldrich, Johannesburg). The extract was centrifuged at 10 000 g for 10 min at 4°C and the supernatant containing the protein was used to determine protein content. Total protein content was determined using the Bradford method with bovine serum albumin (BSA) (Sigma, Aldrich, Johannesburg) as standard (Bradford, 1976). Optical density at 595 nm (OD595) was measured with a BMG FLUOstar Optima microplate reader (Lasec, Cape Town) to determine protein concentration. 2.6. Protease inhibition assay Protease inhibition analysis was modified from the method described by Carrillo et al. (2011). The inhibitory activity of the BBI transgene product in plant protein extracts was performed against commercial trypsin using Z-L-R-AMC (Z-L-Arg-7-Amido-4-methyl-Coumarin) (Sigma, Aldrich, Johannesburg) substrate and the wild-type was used as a control. A final volume of 100 μl containing 20 μg protein extract with 100 ng of trypsin (Sigma, Aldrich, Johannesburg) in TrisHCl 0.1 M, pH 7.5 (Sigma, Aldrich, Johannesburg) was incubated in the dark for 10 min at room temperature in a 96 well micro-plate. The reaction was initiated by adding a 200 μm substrate to the pre-incubated mixture. Fluorescence was measured at an excitation filter of 365 nm and an emission filter of 465 nm at an interval of 30 s for 10 min using a BMG FLUOstar Optima micro-plate reader (Lasec, Cape Town). Proteolytic activity was expressed in fluorescence units per μg of added protein.
2.9. Glutathione-S-transferase (GST) activity Glutathione-S-transferase (GST) activity was measured using the modified protocol from Mannervik et al. (1985). Twenty micro-liters of extracted protein (2 μg) from each sample was aliquoted into plastic cuvettes. To each protein sample, a volume of 980 μl reaction mixture was added containing 1 mM 1-Chloro-2,4-dinitrobenzene (CDNB), 1 mM glutathione made up in 0.05 mM Tris-HCL buffer (pH 6.5) (Merck Millipore, Germiston). Following a 3 min incubation, the absorbance was measured at 340 nm to determine kinetic rate of glutathione-stransferase. The extinction coefficient (ϵ) of 0.0096 μM−1 cm−1 was then used to calculate GST activity in the equation below: 288
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GST activity =
Asample at 3 mins − Asample at 0 mins ε
plants (Fig. 1D) under normal conditions. Both the wild-type and transgenic lines exhibited similar growth characteristics (Fig. 1D).
(3)
3.2. Stress response of transgenic Arabidopsis plants expressing BBI gene to drought: Overexpression of the BBI gene enhanced drought tolerance in Arabidopsis
2.10. Ascorbate peroxidase (APX) activity The modified method of Sign et al. (2007) was used to determine the total ascorbate peroxidase activity. Proteins were extracted and quantified from the frozen ground material. The protein samples were aliquoted into 0.5 ml microfuge tubes and incubated with 50 mM TrisHCl pH 7.5 and 2 mM ascorbate (Merck Millipore, Germiston) for 5 min. The protein samples were then loaded in triplicates in a microtiter plate. To each well containing 20 μg protein sample and blank sample (50 mM Tris-HCl buffer pH 7.5), 10 mM H2O2 was added to start the reaction immediately before taking the absorbance readings at 290 nm on a BioTek PowerWave™ microplate spectrophotometer. Reactions were made up to a total volume of 200 μl. The ascorbate peroxidase activity was measured using an extinction coefficient (ϵ) of 2.8 mM−1. cm−1 in the equation below:
APX activity =
To study the response of BBI-overexpressing Arabidopsis plants to drought stress, progressive drought was conducted. Water was withheld from plants until wilting symptoms were observed in the wild-type. All the wild-type plants exhibited severe water loss related symptoms with severe wilting following drought treatment (Fig. 2A). On the contrary, only slight wilting was observed in the transgenic lines (Fig. 2A) under the same condition. Following water deprivation, Fig. 2B shows that transgenic plants survived better than the wild-type under the same condition. The results indicate a significant decrease (P < 0.05) in the survival rate of the wild-type in drought stress when compared to the well-watered treatment. Relative water content (RWC) was used as a selection criterion for drought tolerance. There were no significant differences (P > 0.05) in the relative water content between the wild-type and the transgenic lines under normal watering. However, under drought conditions, transgenic lines 1 and 2 retained higher leaf water content as compared to transgenic line 3 which was not significantly different from the wildtype. This was evident in the rosette phenotype analysis, where the transgenic plants were visibly turgid at nine days drought treatment (Fig. 2A). The relative water content (Fig. 2C) of the wild-type was significantly decreased (P < 0.0001) to 32% whereas the transgenic lines were decreased to 62.3%, 72.5% & 35.5% (L1 P < 0.05, L2 P < 0.01 and L3 P < 0.001) when comparing the well-watered and drought conditions (WT well-watered vs WT drought). We observed no significant differences (P > 0.05) in the total leaf number of both well-watered and drought-stressed plants (Fig. 2D). The imposed 9-day drought stress resulted in a significant decrease (P < 0.05) of 60% in fresh leaf biomass only in the wild-type plants (Fig. 2E) when compared to the wild-type under well-watered conditions.
Asample at 7 mins − Asample at 0 mins ε
(4)
2.11. Statistical analysis GraphPad prism version 8 software was used for statistical analysis and three independent biological replicates (n = number of replicates used for SEM bars) were subjected to two-way ANOVA using Tukey's multiple comparison post-hoc t-tests (p-value < 0.05) were subsequently performed. 3. Results 3.1. Production of transgenic Arabidopsis overexpressing the maize BBI gene The expression construct containing double CAMV35S promoter driving the BBI gene (Fig. 1A) was transformed using Agrobacteriummediated transformation and three independent transgenic lines (L1, L2 and L3) were obtained. The presence of BBI transgene was detected by PCR (Fig. 1B) on selected plants and the transgenic lines showed the expected 350 bp band which was absent in the wild-type. Homozygous T3 transgenic lines were selected and used for physiological analysis. Analysis with real-time quantitative PCR showed differential expression of BBI transcripts in the three lines (Fig. 1C). The effect of ectopic expression of BBI on vegetative growth was investigated in 5-week old
3.3. Trypsin-inhibitory activity in the BBI-expressing transgenic plants To examine the effect of the ectopic expression of the BBI gene using the trypsin protease inhibitory assay, crude proteins were extracted from all the transgenic lines L1, L2 & L3 and the WT plants (Fig. 3A). For the protease inhibitory assay against trypsin, 20 ng of crude plant protein was tested for trypsin activity against 100 ng trypsin in buffer Fig. 1. Identification of transgenic Arabidopsis plants. (A) The overexpression construct of BBI used for Arabidopsis transformation. (B) PCR analysis of transgenic Arabidopsis overexpressing the BBI gene. (C) Relative expression of BBI in Arabidopsis leaves of transgenic plants. (D) Phenotypic analysis of transgenic plants and wild-type plants. The expression of BBI gene was normalized to αEF, SAND, PDF and FBOX housekeeping genes. Data represent mean n = 3 values ± SEM.
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Fig. 2. Water stress response of BBI transgenic and WT Arabidopsis plants. (A) Four-week-old plants were subjected to progressive drought. (B) The survival rates were determined by counting the number of plants alive following drought stress imposition. (C) The relative water content was measured and compared at 9 days after drought. The leaves of both well-watered and drought-stressed plants were counted (D) and weighed after harvest to determine leaf fresh weight (E). Bars with different letters are significantly different at P < 0.05 (n = 12 ± SEM for survival rate and n = 6 ± SEM for relative water content). Four columns in a block (WT and three transgenic lines) were analyzed for significant differences.
Fig. 3. The effects of BBI transgene expression on leaf protein content (A) and serine protease activity (B). Crude protein was extracted from both well-watered and drought stressed leaves. Serine protease activity was determined by measuring the increase in fluorescence of the substrate at λex 365 nm; λem 465 nm. Bars with the same letters are not significantly different at P < 0.05 (n = 3 ± SEM). Four columns in a block (WT and three transgenic lines) were analyzed for significant differences. 290
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Fig. 4. The effect of drought stress on MDA (A), hydrogen peroxide H2O2, (B) and antioxidant enzyme activity of GST (C) and APX (D) in wild-type and transgenic Arabidopsis plants expressing the BBI gene. Bars with different letters are significantly different at P < 0.05 (n = 3 ± SEM). Four columns in a block (WT and three transgenic lines) were analyzed for significant differences.
drought conditions when compared to well-watered conditions. Under well-watered conditions, transgenic lines APX activity increased to 78%, 79% and 37% for LI, L2 and L3 respectively.
and the results were quantified based on the decrease in the amount of substrate Z-L-R-AMC hydrolyzed by trypsin. The standard reaction with bovine trypsin showed no significant differences (P < 0.05) in trypsin activity. However, L1 and L2 transgenic lines showed a trend of lower trypsin activity under drought stress (Fig. 3B).
4. Discussion Drought stress is one of the major abiotic stresses, which negatively affects plant growth and its effect can be observed at molecular, physiological and morphological level depending on stress severity. In this study we assessed Arabidopsis plants overexpressing a maize serine protease inhibitor, BBI for physiological and biochemical responses to drought stress. Progressive drought, where water is withheld for a certain period until symptoms of wilting occur, was chosen for analysis of abiotic stress response. This method of drought is usually used when determining the survival rate of wild-type plants and monitoring overexpression of genes for drought tolerance (Harb et al., 2010). The BBI gene was successfully transformed into A. thaliana, and this gene's expression was confirmed by analyzing the BBI gene transcript level expression with an RT-qPCR assay. All three transgenic lines showed differential expression of the gene with low accumulation of BBI transcript in line 3 (Fig. 1C). We analyzed how overexpression of this gene affects the phenotype of plants under normal conditions. Unlike the results obtained by Tiwari et al. (2015) where the overexpression of rice chymotrypsin inhibitor gene OCPI2, enhanced vegetative growth in Arabidopsis plants, our transgenic lines showed normal growth with similar measurements of leaf biomass and leaf numbers as the wild-type under normal condition. There is an increasing evidence for the role of serine protease inhibitors in abiotic stress. It was shown that serine and cysteine protease inhibitors accumulate in winter wheat plants under drought stress with one of the serine protease inhibitors being a Bowman-Birk inhibitor (Vaseva et al., 2016). In our study, BBI-expressing plants displayed more tolerance to drought stress when compared to the WT, which showed wilting symptoms at 9 days of water withdrawal while
3.4. Enzymatic activity assays Malondialdehyde (MDA) acts as an indicator of membrane damage and lipid peroxidation during drought. Under drought conditions, the transgenic lines had significantly lower MDA levels when compared to the wild-type plants. Wild-type plants had the highest MDA (P < 0.0001) content increase (52,36%) when compared to the transgenic lines under drought stress (Fig. 4A). These results suggest increased lipid peroxidation in wild-type plants when compared to transgenic lines in water deficit environments. Drought stress often leads to oxidative stress by producing reactive oxygen species such as hydrogen peroxide, which in turn leads to the activation of the antioxidant defense system. Drought elicited a major increase (P < 0.0001) in hydrogen peroxide content, and we observed an 84% increase in H2O2 in the wild-type plants, while transgenic lines increased H2O2 to 82%, 81% and 81% for L1, L2 and L3 respectively (Fig. 4B). In order to validate antioxidant defense system under drought stress, analysis of GST and APX antioxidant enzymes was performed in wild-type and transgenic lines. Glutathione-S-transferase activity (GST) and APX are some of the enzymes involved in detoxifying and protecting plant cells from oxidative stress (Hasanuzzaman et al., 2012). Following drought stress, transgenic plants revealed a significant increase in GST activity (Fig. 4C). Ascorbate peroxidase activity of L1 and L2 was significantly higher than the wild-type under well-watered conditions. The results in Fig. 4D indicate that APX activity was significantly high under well-watered in L1 and L2 plants while the wildtype and L3 showed a significant increase in activity of APX under 291
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These ROS scavenging enzymes have been reported to respond differently under different levels of drought condition (Hameed et al., 2011). Ascorbate peroxidase genes are important for removing H2O2 and reducing cell damage under abiotic stress (Sofo et al., 2015). In our study, transgenic lines 1 and 2 had higher APX content under well-watered conditions as compared to wild-type plants and transgenic line 3. However, there was no significant difference (P > 0.05) in APX activity under drought stress. We observed a significant increase in activity of this enzyme in WT and L3 when comparing well-watered and drought conditions, while L1 and L2 maintained the high activity observed during well-watered conditions. Glutathione acts as a substrate for GSH-metabolism enzymes such as GSTs and glutathione peroxidases. It is considered the most important defense thiol which protects plants from ROS mediated oxidative damage in plants. Glutathione-S-transferase enzymes play a role in phase II GSH dependent ROS-scavenging and they are responsible for detoxifying cells from lipid and protein peroxidation products (Bhardwaj and Yadav, 2012). Previous studies, indicated that GST increases drought tolerance in plants through overexpression of GST (Labudda and SafiulAzam, 2014). In our study Glutathione-S-transferase enzyme was significantly increased in transgenic plants under drought conditions when compared to the wild-type. We therefore suggest that GST enzyme was enhanced in transgenic plants to provide tolerance to drought stress. Drought stress greatly affected Arabidopsis growth and resulted in reduced leaf water potential, high proteolysis and high leaf peroxidation. We successfully expressed the maize BBI gene in Arabidopsis plants, with transgenic lines 1 and 2 showing better performance under drought stress conditions. It is clear from this study that BBI-expressing plants exhibit better performance under drought stress due to the observed reduced increase in antioxidant enzymes activity (GST) and reduced MDA content. The observed results show that this BBI protease inhibitor has regulatory role in the overall protein metabolism under drought stress. In our experiments, there was no significant change in serine protease activity under well-watered and drought treated wildtype and transgenic lines. This can be attributed to the fact that there are numerous serine proteases and we only investigated the trypsin-like proteases. In the future, several other trypsin-like proteases should be investigated using different substrates.
transgenic lines possessed more turgid leaves as a result of the higher RWC observed. Many studies have shown and confirmed that RWC can be used as an indicator for drought tolerance as it signifies the degree of tissue hydration and the ability of plants to retain water use under stressful conditions, which result in the maintenance of physiological functioning and growth processes (Ying et al., 2015). The observed tolerance might be associated with the slight decrease in RWC experienced by transgenic plants while the wild-type maintained lower RWC. Reduction in growth measured as biomass shows either sensitivity or tolerance to drought stress, in our study we noted sensitivity to drought stress in the wild-type compared to the transgenic lines. To understand whether the observed tolerance to drought stress was due to the serine protease inhibitory activity of BBI-expressing plants, protein extracts from transgenic plants were assayed for trypsin activity. Considering the BBI trypsin inhibitor gene is present at high levels in transgenic lines, the activity of BBI was tested against trypsin, under both normal and drought conditions. We noted an increase in protease activity of the wild-type under drought conditions and surprisingly line 3 exhibited similar results as the wild-type. Previous studies have also illustrated higher proteolytic activity in drought sensitive plants (Simova-Stoilova et al., 2009). It is important to note that during drought stress the activity of trypsin in transgenic line 1 and 2 did not change when compared to the well-watered condition, however, under drought condition, a clear trend of decrease in protease activity was observed in transgenic plants when compared to the wild-type under similar conditions. These results suggest possible regulation of endogenous serine proteolysis under drought stress by the BBI gene. In order to evaluate the potential link of drought tolerance observed in transgenic plants to the antioxidant defense system, we characterized the changes in redox and the activation of antioxidant enzyme defense system. It is known that drought stress elicits proteolytic activity and ROS in plants, leading to cellular damage during biotic and abiotic stresses (Hussain et al., 2019). Serine as well as cysteine protease inhibitors have been implicated in the regulation of proteases in order to control proteolysis dependent cellular damage (Dhanushkodi et al., 2018). Lipid peroxidation has also been associated with the activation of ROS, in turn this peroxidation induces the activation of antioxidant defense system in order to maintain the redox homeostasis (You and Chan, 2015). Malondialdehyde is a product of lipid peroxidation and it is used as a molecular marker to estimate the extent of lipid peroxidation in plants (Draper and Hadley, 1990). The content of MDA is known to increase under stress damage, so in general its concentration increases with an increase in drought stress. Our data demonstrate that drought stress induces MDA production, as we observed a significant increase in MDA content of the wild-type plants (wild type well-watered vs wild type drought). On the other hand, transgenic plants displayed significantly lower MDA levels as compared to wild-type under drought conditions, which suggests that these plants experienced less lipid peroxidation. This decrease supports the observed tolerance observed under drought stress condition. Most plant damage during abiotic stress has been linked to oxidative stress at the cellular level where ROS like H2O2 may cause damage to membrane lipids, leading to cell death (Hameed et al., 2011). Hydrogen peroxide is involved in signaling which activates multiple defense responses leading to the enhancement of abiotic stress tolerance (Hossain et al., 2015). The results obtained in our study indicate that ROS was activated in the wild-type and transgenic plants as a result of the imposed drought stress. A dramatic increase in H2O2 was observed under drought stress for both the wild-type as well as the transgenic lines. We further investigated the induction of the antioxidant defense system by evaluating the activity of APX and GST antioxidant enzymes, which form part of the enzymatic components of the antioxidant defense system. Antioxidant enzymes such as ascorbate peroxidase (APX) and catalase (CAT) play a role in maintaining the H2O2 homeostasis in plants.
5. Contributions Malefo MB carried out experiments of most of the drought experiments and in the manuscript preparation. Mathibela EO carried out the antioxidant experiments. Crampton BG supervised and helped in the preparation of the manuscript. Makgopa ME prepared the constructs for transformation, carried out the transformation and was involved in the manuscript preparation. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported in part by the National research foundation (NRF) [grant numbers 113591, 2018]. Authors E.O. Mathibela and M. B. Malefo are grateful to NRF and University of Pretoria, for masters and doctoral scholarship; respectively. References Ahmad, P., Abdel Latef, A.A.H., Rasool, S., Akram, N.A., Ashraf, M., Gucel, S., 2016. Role of proteomics in crop stress tolerance. Front. Plant Sci. 7.
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Investigating the role of Bowman-Birk serine protease inhibitor in Arabidopsis plants under drought stress
T
M.B. Malefoa,b, E.O. Mathibelaa,b, B.G. Cramptona,b, M.E. Makgopaa,b,∗ a b
Department of Plant and Soil Sciences, Private Bag X20, Hatfield, Pretoria, 0001, South Africa Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, Hatfield, 0028, South Africa
A R T I C LE I N FO
A B S T R A C T
Keywords: Bowman-birk inhibitor Serine protease inhibitor Drought tolerance Arabidopsis Antioxidant enzymes
Serine protease inhibitors (SPIs) play an important role in cell survival, development and host defense. In plants, serine protease inhibitors such as the Kunitz-type inhibitor (KTI) and the Bowman-Birk inhibitor (BBI) have been shown to be induced in response to abiotic stress such as salinity and drought resulting in tolerance to these stresses. In this study, Arabidopsis thaliana (T3) plants overexpressing the BBI gene from maize were generated and subjected to drought stress in order to study the role of BBI protease inhibitor in drought tolerance. Drought treatment of four-week-old Arabidopsis plants was performed by withholding water from plants for nine days and harvested plant material was used for physiological and biochemical analysis. The transgenic lines exhibited normal growth after nine days of drought as compared to the wild-type. The results also showed a higher leaf relative water content (RWC) in transgenic lines when compared to the wild-type (WT), with line 2 having the highest RWC of 72% and the WT having the lowest RWC of 32%. Trypsin-inhibitor activity indicated that the total protein of the positive transgenic plants had stronger protease inhibitory activity than the wild-type. Transgenic lines overexpressing BBI also showed reduced lipid peroxidation (MDA content) as well as enhanced activity of antioxidants glutathione-s-transferase (GST) and ascorbate peroxidase (APX). These results suggest that BBI protease inhibitor leads to drought tolerance associated with reduction in drought-induced oxidative stress.
1. Introduction Plants are often exposed to abiotic stresses such as drought, salinity, cold, heat and extreme light which pose a threat to plant production and growth. These abiotic stresses are responsible for crop losses worldwide, resulting in yield reductions of over 50% (Ahmad et al., 2016). Availability of water is one of the key factors that contribute to the reduction of crop production. Drought is thus one of the mostly studied abiotic stresses as it severely affects the growth and development of plants. The effect of drought in plants is observed at the cellular level, where it disrupts cellular functions (Bray, 2001), and it also affects the photosynthetic parts of the plant, which results in reduced photosynthetic rates, destroyed chloroplast structures and restrictions to electron transport and enzyme activity (Zhou et al., 2015). The study of variations in gene expression during drought is commonly used to resolve the molecular basis of drought stress leading to either susceptibility or tolerance to drought. For many years
Arabidopsis has been used as a model plant, linking cellular responses and gene expression data. Together with soybean and tobacco, Arabidopsis has been used for the improvement of water use efficiency in crops and the regulatory genes underlying the efficiency have been studied in these plants (Zhou et al., 2015). Studies have shown that post-translational regulation can result in poor correlations between transcripts and their proteins in the transcriptomic analysis of samples under drought conditions and thus, proteomics can be used as a powerful tool in exploring the response mechanisms of plants to various stresses (Ali and Komatsu, 2006). Protein breakdown has been identified as one of the key adaptations of plants to abiotic stress (Budič et al., 2016). In plants, a group of proteolytic enzymes called proteases are responsible for protein breakdown. Proteases hydrolyze specific peptide bonds in proteins, and they are classified based on the type of active site residue they possess i.e. cysteine, metallo-, aspartic, glutamic, asparagine, and serine proteases (Barrett, 1986). These proteolytic enzymes are responsible for
Abbreviations: RWC, relative water content; BBI, Bowman-Birk inhibitor; SPI, serine protease inhibitor; PI, protease inhibitor; GST, glutathione-S transferase; APX, ascorbate peroxidase; H2O2, hydrogen peroxide; MDA, malondialdehyde ∗ Corresponding author. Department of Plant and Soil Sciences, Private Bag X20, Hatfield, Pretoria, 0001, South Africa. E-mail address: [email protected] (M.E. Makgopa). https://doi.org/10.1016/j.plaphy.2020.02.007 Received 19 October 2019; Received in revised form 8 February 2020; Accepted 9 February 2020 Available online 15 February 2020 0981-9428/ © 2020 Elsevier Masson SAS. All rights reserved.
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resistance to the herbicide phosphinothricin and its derivatives. Agrobacterium-mediated transformation was performed according to the floral dip method (Clough and Bent, 1998). Wild-type A. thaliana (ecotype Col-0) plants were grown in controlled environment chambers, at day/night temperatures of 25°C/20°C and an irradiance of 400 μmol m−2s−1 with a 16h photoperiod. Primary inflorescence buds were clipped to allow the formation of secondary inflorescence buds and increase the transformation process by obtaining more flower buds per plant. Plants were grown for 8 weeks before floral dip transformation with Agrobacterium tumefaciens strain GV3101 carrying the plasmid pTF101.1-BBI (Clough and Bent, 1998). Secondary inflorescences were submerged into the A. tumefaciens cell suspension for 10 s and then allowed to grow in the controlled environment chambers until seed maturity.
cellular housekeeping and stress response by degrading misfolded and potentially harmful proteins, to provide amino acids for the production of new proteins (Gur et al., 2011). Proteases play a major role in protein breakdown during leaf senescence under biotic and abiotic stresses. Proteolytic activity also increases during drought stress as a result of hydrogen peroxide production leading to the enhancement of leaf senescence and drought stress sensitivity (Dhanushkodi et al., 2018). Studies by Chen et al. (2013), reported enhanced drought sensitivity in transgenic lines overexpressing the sweet potato cysteine protease gene, SPCP3. Evidence of papain-like cysteine proteases that lead to oxidative damage and programmed cell death (PCD) have been previously reported, indicating the involvement of proteases in abiotic stress susceptibility (Lampl et al., 2013). Serine proteases are the most prominent of the seven families of proteases in plants. Serine proteases catalyze the hydrolysis of specific peptide bonds in their substrate, which contain a serine amino acid residue in the active site. This family of proteases is involved in many physiological processes which includes the hypersensitive response, signal transduction, senescence and protein degradation (Asif-Ullah et al., 2006). A recent study by Fan et al. (2016) also suggests that serine protease expression could be involved in defense response against biotic and abiotic stresses. Despite being important for cellular functions, proteases can be very damaging when overexpressed, leading to degradation of many proteins in response to environmental factors and as a result, they are strictly regulated. Cell damage by proteases leads to the expression of protease inhibitors (PI) which are used to control the unwanted proteolysis by proteases (Yang and Yeh, 2005). Protease inhibitors are plant proteins which function by regulating proteolytic activity of their target proteases, resulting in the formation of a stable protease inhibitor complex (Meenu and Murugan, 2016). Protease inhibitors have also been shown to play an important role in abiotic stress tolerance. In Arabidopsis thaliana, cysteine protease inhibitors were noted to increase salt, drought, oxidation and cold tolerance (Zhang, 2008). Serine protease inhibitors are well-known defense components in plants, and they are responsible for inhibiting trypsin and chymotrypsin. Plant inhibitors such as the Kunitz-type inhibitor (KTI) and Bowman-Birk inhibitor (BBI) genes, encoding serine protease inhibitors have been shown to play a role in plant defense against insects and pathogens (Dramé et al., 2013). The induction of BBIs has been observed during abiotic stresses such as salinity and metal toxicity. In maize, BBI gene transcript expression was previously found to be induced by high carbon dioxide concentration (Prins et al., 2011). However, the exact role of this protease inhibitor remains unknown. Although drought is one of the well-studied stresses, the involvement of protease inhibitors in enhanced tolerance has not been thoroughly studied. In this study, the BBI gene was overexpressed in Arabidopsis thaliana and its effect on improving drought tolerance in transgenic A. thaliana was evaluated.
2.2. Selection of transgenic lines 2.2.1. Plant growth and maintenance Transgenic T0 Arabidopsis seeds were sterilized in 70% ethanol for 5 min and in 10% bleach containing 0.1% Triton X for 15 min then washed with sterile water three times and re-suspended in 0.1% agar and placed on MS medium containing 50 mg/l Basta®. The plates were incubated at 4°C for 2 days in the dark and grown in constant light at 25°C for 7 days. Seedlings were transferred to moistened peat pellets (Jiffy product) and maintained until they were four weeks old. The plants were grown to maturity in a controlled growth room, at full day conditions of 16 hr light/8 hr day photoperiod at a temperature of 20–23°C and humidity of 45% RH. The following studies were performed on three independent transformed homozygous lines (Line 1, Line 2, and Line 3) and the wild-type (WT). 2.2.2. PCR amplification of the maize BBI gene Amplification of the BBI was performed using the Bio-Rad S1000™ Thermal Cycler (Bio-Rad Laboratories, California). A PCR product of 350 bp was amplified using maize cDNA specific BBI primers; forward primer 5′-GCC AGG ACA GGA GAA ACA AA-3′ and reverse primer 5′CAT GCC GTA CGT CAG AAG AA-3’. PCR was performed at 30 cycles using a 50 ng gDNA template per reaction in a PCR tube with DreamTaq PCR Master Mix (2X) (ThermoFisher Scientific, Johannesburg) in 25 μl reaction volume containing 0.5 μM forward and 0.5 μM reverse primers. PCR conditions were 1 cycle denaturation step at 94°C for 3 min; 30 cycles denaturation/annealing/elongation at 94°C for 1 min/60°C for 1 min/72°C for 1 min followed by 1 cycle final extension at 72°C for 10 min. The PCR products were verified by agarose gel electrophoresis analysis and then used for sequencing to confirm the BBI sequence (Sanger et al., 1977). 2.2.3. RNA extraction and RT-qPCR analysis Total RNA was extracted from the fully expanded leaves by ZymoBIOMICS™ RNA Mini Kit (Inqaba Technical industries, Pretoria) following the manufacturer's instructions. The RNA concentration was calculated from the optical density (OD) of the purified RNA at 260 nm wavelength using the Nano-Drop™ spectrophotometer (ThermoScientific, Waltham, Massachusetts) and 1 μg of RNA was transcribed to cDNA using the ReverseAid first strand cDNA synthesis kit transcriptase (ThermoFisher Scientific, Johannesburg). Real-time PCR was performed for the BBI gene using the gene-specific primers: 5′CAT CCG GAG TGC ATG AAC T-3′ and 5′-GTT TGT GAG AAC GTC GTC AC-3’. RT-qPCR assays were performed using a CFX96 Touch™ RealTime PCR Detection System in a 96-well Clear Multiplate PCR Plates (Bio-Rad, California). Each 10 μl reaction comprised 1 μl cDNA template, 5 μL KAPA SYBR® FAST qPCR Master Mix (Lasec SA (Pty) Ltd), Cape Town, 0.2 μM of each primer and 4 μL of sterile distilled water. For the non-template control, cDNA was replaced with sterile distilled water. The reactions were subjected to an initial denaturation step at 95°C during 10 min followed by 45 cycles at 95°C for 10 s, 60°C for 30 s
2. Materials and methods 2.1. Constructs and A. thaliana transformation The BBI gene (Accession number EF406276.1) was amplified from maize (Zea may L.) cDNA with gene-specific primers containing restriction enzyme sites BamHI and HindIII; BBI-HindIII forward primer 5′CCCG AAG CTT GCC AGG ACA GGA GAA ACA AA-3′ and BBI-BamHI reverse primer 5′-TCCT GGA TCC CAT GCC GTA CGT CAG AAG AA-3’. The amplicon was purified and cloned into pLBR 19 plasmid. The expression cassette of plasmid pLBR-BBI was sub-cloned into the SacI HF™ and XbaI restriction sites of plasmid pTF101.1 containing a spectinomycin resistant marker gene (aaadA) for bacterial selection. The plant expression cassette used contained the cauliflower mosaic virus (CaMV) double 35S promoter (2 × 35S) and the phosphine acetyltransferase gene (bar gene) from Streptomyces hygroscopicus which conferred 287
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2.7. Determination of lipid peroxidation
and 72°C for 15 s. The transcript level of BBI gene was estimated relative to four Arabidopsis reference genes; EFα: 5′-CAC CAC TGG AGG TTT TGA GG-3′ and 5′-TGG AGT ATT TGG GGG TGG T-3’ (GenBank accession At5g60390), SAND 5′-GTT GGG TCA CAC CAG ATT TTG and 5′-GCT CCT TGC AAG AAC ACT TCA-3, (GenBank accession At2g28390), PDF2 5′-TCA TTC CGA TAG TCG ACC AAG and 5′-TTG ATT TGC GAA ATA CCG AAC-3’ (GenBank accession At1g13320) and FBOX 5′-GGC TGA GAG GTT CGA GTG TT-3′ and 5′-GGC TGT TGC ATG ACT GAA GA-3’ (GenBank accession At5g15710). Relative gene expression levels were calculated using the 2−ΔΔCT method with three independent biological replicates (Livak and Schmittgen, 2001).
Lipid peroxidation was determined as previously described by Zhang et al. (2007). Whole Arabidopsis rosettes (100–700 mg) were ground in liquid nitrogen and placed into microfuge tube. Five volumes of 6% (w/v) trichloroacetic acid (TCA) (Merck Millipore, Germiston) was added to each microfuge tubes. The samples were homogenized using a vortex and then centrifuged at 10 000 g for 10 min to pellet the plant material. Approximately 200 μl of supernatant was transferred to a new microfuge tube followed by the addition of 300 μl of 0.5% (w/v) thiobarbituric acid (TBA) (Sigma, Aldrich, Johannesburg). The samples were mixed by brief vortexing. A small hole was punched on the microfuge lids to prevent the lids from opening under high temperature incubation. The sample tubes were placed in a heating block and were allowed to incubate at 90°C for 20 min. Following the 20 min incubation, the samples were incubated on ice for 10 min. Once the incubation was complete, the samples were centrifuged at 10 000 g for 5 min to pellet plant material. All extracted samples were then loaded in triplicate on a 96-well micro-titer plate. The plate was then read at 532 nm and 600 nm on a PowerWave™ microplate spectrophotometer (BioTek® instruments, INC, Winooski). The absorbance at 600 nm was subtracted from the absorbance at 532 nm to correct for the non-specific turbidity. Lipid peroxidation was determined by measuring activity of malondialdehyde (MDA) using spectrophotometry applying the extinction coefficient 155 Mm cm−1. Percentage increase was calculated by the equation:
2.3. Drought tolerance analysis of transgenic plants Seedlings (T3) of wild-type and transgenic plants (approximately 30 of each line) were germinated on moistened peat pellets (Jiffy product) and grown under short-day conditions (8 h light/16 h dark) at 22–24°C with normal watering for four weeks. The four-week-old plants were subjected to drought stress by withholding water for nine days thereafter, photos were taken, and plants were harvested for RNA and protein extractions. The whole rosette was harvested and then weighed for protein measurements. 2.4. Determination of relative water content For the quantification of relative water content, the whole rosette was collected from both well-watered and drought-treated plants and immediately weighed for fresh weight. The leaf material was then placed at 80°C overnight then weighed the samples for dry weight measurements. The relative water content was calculated by the equation (Quain et al., 2014):
RWC % =
fresh weight − dry weight X 100 fresh weight
%change =
Xwell − watered − Xdrought Xwell − watered
X 100
(2)
2.8. Hydrogen peroxide (H2O2) content A modified method of Velikova et al. (2000) was followed to determine the H2O2 content in the plant material. Whole Arabidopsis rosettes (100–700 mg) were ground in liquid nitrogen and inserted in a microfuge tube. Five volumes of 6% (w/v) trichloroacetic acid (TCA) (Merck Millipore, Germiston) was added to the microfuge tubes. The samples were homogenized using a vortex and then centrifuged at 10 000 g for 10 min to pellet the plant material. Approximately 200 μl of supernatant was transferred to a new microfuge tube followed by the addition of 300 μl of 0.5% (w/v) thiobarbituric acid (TBA) (Sigma, Aldrich, Johannesburg). The samples were mixed by brief vortexing. Standards from a 30% H2O2 stock (Sigma Aldrich, Johannesburg) ranging from 10, 20, 30, 40, 50, 60, 70, 80, and 100 μM were prepared by diluting an appropriate volume of H2O2 in deionised water and used to measure H2O2 in extracted samples. A volume of 50 μl extracted sample and standards were then loaded in triplicate on a 96-well microtiter plate. To the samples and standards, 1.25 mM dipotassium hydrogen phosphate (K2HPO4) and 250 mM potassium iodide (KI) were added. Once the plate was prepared and the solutions properly mixed, it was incubated for 20 min at room temperature. The samples were then read at 390 nm using a PowerWave™ microplate spectrophotometer (BioTek® instruments, INC, Winooski).
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2.5. Protein extraction and yield determination Total protein extracts were obtained from leaf rosette of transgenic and wild-type Arabidopsis plants. The leaves (100 mg) were ground into a powder and dissolved in 50 mM Tris-HCl, pH 8 (Sigma, Aldrich, Johannesburg). The extract was centrifuged at 10 000 g for 10 min at 4°C and the supernatant containing the protein was used to determine protein content. Total protein content was determined using the Bradford method with bovine serum albumin (BSA) (Sigma, Aldrich, Johannesburg) as standard (Bradford, 1976). Optical density at 595 nm (OD595) was measured with a BMG FLUOstar Optima microplate reader (Lasec, Cape Town) to determine protein concentration. 2.6. Protease inhibition assay Protease inhibition analysis was modified from the method described by Carrillo et al. (2011). The inhibitory activity of the BBI transgene product in plant protein extracts was performed against commercial trypsin using Z-L-R-AMC (Z-L-Arg-7-Amido-4-methyl-Coumarin) (Sigma, Aldrich, Johannesburg) substrate and the wild-type was used as a control. A final volume of 100 μl containing 20 μg protein extract with 100 ng of trypsin (Sigma, Aldrich, Johannesburg) in TrisHCl 0.1 M, pH 7.5 (Sigma, Aldrich, Johannesburg) was incubated in the dark for 10 min at room temperature in a 96 well micro-plate. The reaction was initiated by adding a 200 μm substrate to the pre-incubated mixture. Fluorescence was measured at an excitation filter of 365 nm and an emission filter of 465 nm at an interval of 30 s for 10 min using a BMG FLUOstar Optima micro-plate reader (Lasec, Cape Town). Proteolytic activity was expressed in fluorescence units per μg of added protein.
2.9. Glutathione-S-transferase (GST) activity Glutathione-S-transferase (GST) activity was measured using the modified protocol from Mannervik et al. (1985). Twenty micro-liters of extracted protein (2 μg) from each sample was aliquoted into plastic cuvettes. To each protein sample, a volume of 980 μl reaction mixture was added containing 1 mM 1-Chloro-2,4-dinitrobenzene (CDNB), 1 mM glutathione made up in 0.05 mM Tris-HCL buffer (pH 6.5) (Merck Millipore, Germiston). Following a 3 min incubation, the absorbance was measured at 340 nm to determine kinetic rate of glutathione-stransferase. The extinction coefficient (ϵ) of 0.0096 μM−1 cm−1 was then used to calculate GST activity in the equation below: 288
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GST activity =
Asample at 3 mins − Asample at 0 mins ε
plants (Fig. 1D) under normal conditions. Both the wild-type and transgenic lines exhibited similar growth characteristics (Fig. 1D).
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3.2. Stress response of transgenic Arabidopsis plants expressing BBI gene to drought: Overexpression of the BBI gene enhanced drought tolerance in Arabidopsis
2.10. Ascorbate peroxidase (APX) activity The modified method of Sign et al. (2007) was used to determine the total ascorbate peroxidase activity. Proteins were extracted and quantified from the frozen ground material. The protein samples were aliquoted into 0.5 ml microfuge tubes and incubated with 50 mM TrisHCl pH 7.5 and 2 mM ascorbate (Merck Millipore, Germiston) for 5 min. The protein samples were then loaded in triplicates in a microtiter plate. To each well containing 20 μg protein sample and blank sample (50 mM Tris-HCl buffer pH 7.5), 10 mM H2O2 was added to start the reaction immediately before taking the absorbance readings at 290 nm on a BioTek PowerWave™ microplate spectrophotometer. Reactions were made up to a total volume of 200 μl. The ascorbate peroxidase activity was measured using an extinction coefficient (ϵ) of 2.8 mM−1. cm−1 in the equation below:
APX activity =
To study the response of BBI-overexpressing Arabidopsis plants to drought stress, progressive drought was conducted. Water was withheld from plants until wilting symptoms were observed in the wild-type. All the wild-type plants exhibited severe water loss related symptoms with severe wilting following drought treatment (Fig. 2A). On the contrary, only slight wilting was observed in the transgenic lines (Fig. 2A) under the same condition. Following water deprivation, Fig. 2B shows that transgenic plants survived better than the wild-type under the same condition. The results indicate a significant decrease (P < 0.05) in the survival rate of the wild-type in drought stress when compared to the well-watered treatment. Relative water content (RWC) was used as a selection criterion for drought tolerance. There were no significant differences (P > 0.05) in the relative water content between the wild-type and the transgenic lines under normal watering. However, under drought conditions, transgenic lines 1 and 2 retained higher leaf water content as compared to transgenic line 3 which was not significantly different from the wildtype. This was evident in the rosette phenotype analysis, where the transgenic plants were visibly turgid at nine days drought treatment (Fig. 2A). The relative water content (Fig. 2C) of the wild-type was significantly decreased (P < 0.0001) to 32% whereas the transgenic lines were decreased to 62.3%, 72.5% & 35.5% (L1 P < 0.05, L2 P < 0.01 and L3 P < 0.001) when comparing the well-watered and drought conditions (WT well-watered vs WT drought). We observed no significant differences (P > 0.05) in the total leaf number of both well-watered and drought-stressed plants (Fig. 2D). The imposed 9-day drought stress resulted in a significant decrease (P < 0.05) of 60% in fresh leaf biomass only in the wild-type plants (Fig. 2E) when compared to the wild-type under well-watered conditions.
Asample at 7 mins − Asample at 0 mins ε
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2.11. Statistical analysis GraphPad prism version 8 software was used for statistical analysis and three independent biological replicates (n = number of replicates used for SEM bars) were subjected to two-way ANOVA using Tukey's multiple comparison post-hoc t-tests (p-value < 0.05) were subsequently performed. 3. Results 3.1. Production of transgenic Arabidopsis overexpressing the maize BBI gene The expression construct containing double CAMV35S promoter driving the BBI gene (Fig. 1A) was transformed using Agrobacteriummediated transformation and three independent transgenic lines (L1, L2 and L3) were obtained. The presence of BBI transgene was detected by PCR (Fig. 1B) on selected plants and the transgenic lines showed the expected 350 bp band which was absent in the wild-type. Homozygous T3 transgenic lines were selected and used for physiological analysis. Analysis with real-time quantitative PCR showed differential expression of BBI transcripts in the three lines (Fig. 1C). The effect of ectopic expression of BBI on vegetative growth was investigated in 5-week old
3.3. Trypsin-inhibitory activity in the BBI-expressing transgenic plants To examine the effect of the ectopic expression of the BBI gene using the trypsin protease inhibitory assay, crude proteins were extracted from all the transgenic lines L1, L2 & L3 and the WT plants (Fig. 3A). For the protease inhibitory assay against trypsin, 20 ng of crude plant protein was tested for trypsin activity against 100 ng trypsin in buffer Fig. 1. Identification of transgenic Arabidopsis plants. (A) The overexpression construct of BBI used for Arabidopsis transformation. (B) PCR analysis of transgenic Arabidopsis overexpressing the BBI gene. (C) Relative expression of BBI in Arabidopsis leaves of transgenic plants. (D) Phenotypic analysis of transgenic plants and wild-type plants. The expression of BBI gene was normalized to αEF, SAND, PDF and FBOX housekeeping genes. Data represent mean n = 3 values ± SEM.
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Fig. 2. Water stress response of BBI transgenic and WT Arabidopsis plants. (A) Four-week-old plants were subjected to progressive drought. (B) The survival rates were determined by counting the number of plants alive following drought stress imposition. (C) The relative water content was measured and compared at 9 days after drought. The leaves of both well-watered and drought-stressed plants were counted (D) and weighed after harvest to determine leaf fresh weight (E). Bars with different letters are significantly different at P < 0.05 (n = 12 ± SEM for survival rate and n = 6 ± SEM for relative water content). Four columns in a block (WT and three transgenic lines) were analyzed for significant differences.
Fig. 3. The effects of BBI transgene expression on leaf protein content (A) and serine protease activity (B). Crude protein was extracted from both well-watered and drought stressed leaves. Serine protease activity was determined by measuring the increase in fluorescence of the substrate at λex 365 nm; λem 465 nm. Bars with the same letters are not significantly different at P < 0.05 (n = 3 ± SEM). Four columns in a block (WT and three transgenic lines) were analyzed for significant differences. 290
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Fig. 4. The effect of drought stress on MDA (A), hydrogen peroxide H2O2, (B) and antioxidant enzyme activity of GST (C) and APX (D) in wild-type and transgenic Arabidopsis plants expressing the BBI gene. Bars with different letters are significantly different at P < 0.05 (n = 3 ± SEM). Four columns in a block (WT and three transgenic lines) were analyzed for significant differences.
drought conditions when compared to well-watered conditions. Under well-watered conditions, transgenic lines APX activity increased to 78%, 79% and 37% for LI, L2 and L3 respectively.
and the results were quantified based on the decrease in the amount of substrate Z-L-R-AMC hydrolyzed by trypsin. The standard reaction with bovine trypsin showed no significant differences (P < 0.05) in trypsin activity. However, L1 and L2 transgenic lines showed a trend of lower trypsin activity under drought stress (Fig. 3B).
4. Discussion Drought stress is one of the major abiotic stresses, which negatively affects plant growth and its effect can be observed at molecular, physiological and morphological level depending on stress severity. In this study we assessed Arabidopsis plants overexpressing a maize serine protease inhibitor, BBI for physiological and biochemical responses to drought stress. Progressive drought, where water is withheld for a certain period until symptoms of wilting occur, was chosen for analysis of abiotic stress response. This method of drought is usually used when determining the survival rate of wild-type plants and monitoring overexpression of genes for drought tolerance (Harb et al., 2010). The BBI gene was successfully transformed into A. thaliana, and this gene's expression was confirmed by analyzing the BBI gene transcript level expression with an RT-qPCR assay. All three transgenic lines showed differential expression of the gene with low accumulation of BBI transcript in line 3 (Fig. 1C). We analyzed how overexpression of this gene affects the phenotype of plants under normal conditions. Unlike the results obtained by Tiwari et al. (2015) where the overexpression of rice chymotrypsin inhibitor gene OCPI2, enhanced vegetative growth in Arabidopsis plants, our transgenic lines showed normal growth with similar measurements of leaf biomass and leaf numbers as the wild-type under normal condition. There is an increasing evidence for the role of serine protease inhibitors in abiotic stress. It was shown that serine and cysteine protease inhibitors accumulate in winter wheat plants under drought stress with one of the serine protease inhibitors being a Bowman-Birk inhibitor (Vaseva et al., 2016). In our study, BBI-expressing plants displayed more tolerance to drought stress when compared to the WT, which showed wilting symptoms at 9 days of water withdrawal while
3.4. Enzymatic activity assays Malondialdehyde (MDA) acts as an indicator of membrane damage and lipid peroxidation during drought. Under drought conditions, the transgenic lines had significantly lower MDA levels when compared to the wild-type plants. Wild-type plants had the highest MDA (P < 0.0001) content increase (52,36%) when compared to the transgenic lines under drought stress (Fig. 4A). These results suggest increased lipid peroxidation in wild-type plants when compared to transgenic lines in water deficit environments. Drought stress often leads to oxidative stress by producing reactive oxygen species such as hydrogen peroxide, which in turn leads to the activation of the antioxidant defense system. Drought elicited a major increase (P < 0.0001) in hydrogen peroxide content, and we observed an 84% increase in H2O2 in the wild-type plants, while transgenic lines increased H2O2 to 82%, 81% and 81% for L1, L2 and L3 respectively (Fig. 4B). In order to validate antioxidant defense system under drought stress, analysis of GST and APX antioxidant enzymes was performed in wild-type and transgenic lines. Glutathione-S-transferase activity (GST) and APX are some of the enzymes involved in detoxifying and protecting plant cells from oxidative stress (Hasanuzzaman et al., 2012). Following drought stress, transgenic plants revealed a significant increase in GST activity (Fig. 4C). Ascorbate peroxidase activity of L1 and L2 was significantly higher than the wild-type under well-watered conditions. The results in Fig. 4D indicate that APX activity was significantly high under well-watered in L1 and L2 plants while the wildtype and L3 showed a significant increase in activity of APX under 291
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These ROS scavenging enzymes have been reported to respond differently under different levels of drought condition (Hameed et al., 2011). Ascorbate peroxidase genes are important for removing H2O2 and reducing cell damage under abiotic stress (Sofo et al., 2015). In our study, transgenic lines 1 and 2 had higher APX content under well-watered conditions as compared to wild-type plants and transgenic line 3. However, there was no significant difference (P > 0.05) in APX activity under drought stress. We observed a significant increase in activity of this enzyme in WT and L3 when comparing well-watered and drought conditions, while L1 and L2 maintained the high activity observed during well-watered conditions. Glutathione acts as a substrate for GSH-metabolism enzymes such as GSTs and glutathione peroxidases. It is considered the most important defense thiol which protects plants from ROS mediated oxidative damage in plants. Glutathione-S-transferase enzymes play a role in phase II GSH dependent ROS-scavenging and they are responsible for detoxifying cells from lipid and protein peroxidation products (Bhardwaj and Yadav, 2012). Previous studies, indicated that GST increases drought tolerance in plants through overexpression of GST (Labudda and SafiulAzam, 2014). In our study Glutathione-S-transferase enzyme was significantly increased in transgenic plants under drought conditions when compared to the wild-type. We therefore suggest that GST enzyme was enhanced in transgenic plants to provide tolerance to drought stress. Drought stress greatly affected Arabidopsis growth and resulted in reduced leaf water potential, high proteolysis and high leaf peroxidation. We successfully expressed the maize BBI gene in Arabidopsis plants, with transgenic lines 1 and 2 showing better performance under drought stress conditions. It is clear from this study that BBI-expressing plants exhibit better performance under drought stress due to the observed reduced increase in antioxidant enzymes activity (GST) and reduced MDA content. The observed results show that this BBI protease inhibitor has regulatory role in the overall protein metabolism under drought stress. In our experiments, there was no significant change in serine protease activity under well-watered and drought treated wildtype and transgenic lines. This can be attributed to the fact that there are numerous serine proteases and we only investigated the trypsin-like proteases. In the future, several other trypsin-like proteases should be investigated using different substrates.
transgenic lines possessed more turgid leaves as a result of the higher RWC observed. Many studies have shown and confirmed that RWC can be used as an indicator for drought tolerance as it signifies the degree of tissue hydration and the ability of plants to retain water use under stressful conditions, which result in the maintenance of physiological functioning and growth processes (Ying et al., 2015). The observed tolerance might be associated with the slight decrease in RWC experienced by transgenic plants while the wild-type maintained lower RWC. Reduction in growth measured as biomass shows either sensitivity or tolerance to drought stress, in our study we noted sensitivity to drought stress in the wild-type compared to the transgenic lines. To understand whether the observed tolerance to drought stress was due to the serine protease inhibitory activity of BBI-expressing plants, protein extracts from transgenic plants were assayed for trypsin activity. Considering the BBI trypsin inhibitor gene is present at high levels in transgenic lines, the activity of BBI was tested against trypsin, under both normal and drought conditions. We noted an increase in protease activity of the wild-type under drought conditions and surprisingly line 3 exhibited similar results as the wild-type. Previous studies have also illustrated higher proteolytic activity in drought sensitive plants (Simova-Stoilova et al., 2009). It is important to note that during drought stress the activity of trypsin in transgenic line 1 and 2 did not change when compared to the well-watered condition, however, under drought condition, a clear trend of decrease in protease activity was observed in transgenic plants when compared to the wild-type under similar conditions. These results suggest possible regulation of endogenous serine proteolysis under drought stress by the BBI gene. In order to evaluate the potential link of drought tolerance observed in transgenic plants to the antioxidant defense system, we characterized the changes in redox and the activation of antioxidant enzyme defense system. It is known that drought stress elicits proteolytic activity and ROS in plants, leading to cellular damage during biotic and abiotic stresses (Hussain et al., 2019). Serine as well as cysteine protease inhibitors have been implicated in the regulation of proteases in order to control proteolysis dependent cellular damage (Dhanushkodi et al., 2018). Lipid peroxidation has also been associated with the activation of ROS, in turn this peroxidation induces the activation of antioxidant defense system in order to maintain the redox homeostasis (You and Chan, 2015). Malondialdehyde is a product of lipid peroxidation and it is used as a molecular marker to estimate the extent of lipid peroxidation in plants (Draper and Hadley, 1990). The content of MDA is known to increase under stress damage, so in general its concentration increases with an increase in drought stress. Our data demonstrate that drought stress induces MDA production, as we observed a significant increase in MDA content of the wild-type plants (wild type well-watered vs wild type drought). On the other hand, transgenic plants displayed significantly lower MDA levels as compared to wild-type under drought conditions, which suggests that these plants experienced less lipid peroxidation. This decrease supports the observed tolerance observed under drought stress condition. Most plant damage during abiotic stress has been linked to oxidative stress at the cellular level where ROS like H2O2 may cause damage to membrane lipids, leading to cell death (Hameed et al., 2011). Hydrogen peroxide is involved in signaling which activates multiple defense responses leading to the enhancement of abiotic stress tolerance (Hossain et al., 2015). The results obtained in our study indicate that ROS was activated in the wild-type and transgenic plants as a result of the imposed drought stress. A dramatic increase in H2O2 was observed under drought stress for both the wild-type as well as the transgenic lines. We further investigated the induction of the antioxidant defense system by evaluating the activity of APX and GST antioxidant enzymes, which form part of the enzymatic components of the antioxidant defense system. Antioxidant enzymes such as ascorbate peroxidase (APX) and catalase (CAT) play a role in maintaining the H2O2 homeostasis in plants.
5. Contributions Malefo MB carried out experiments of most of the drought experiments and in the manuscript preparation. Mathibela EO carried out the antioxidant experiments. Crampton BG supervised and helped in the preparation of the manuscript. Makgopa ME prepared the constructs for transformation, carried out the transformation and was involved in the manuscript preparation. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported in part by the National research foundation (NRF) [grant numbers 113591, 2018]. Authors E.O. Mathibela and M. B. Malefo are grateful to NRF and University of Pretoria, for masters and doctoral scholarship; respectively. References Ahmad, P., Abdel Latef, A.A.H., Rasool, S., Akram, N.A., Ashraf, M., Gucel, S., 2016. Role of proteomics in crop stress tolerance. Front. Plant Sci. 7.
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