Plant Physiology and Biochemistry 92 (2015) 48e55
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants Lalit Dev Tiwari, Dheeraj Mittal, Ratnesh Chandra Mishra, Anil Grover* Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 February 2015 Accepted 31 March 2015 Available online 16 April 2015
Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Arabidopsis Chymotrypsin protease inhibitor Growth Osmotic stress Rice Salt stress
1. Introduction Along with proteases and their substrate proteins, protease inhibitors (PI) constitute an important component of the protein turnover machinery. PIs keep a check on the otherwise damaging effects of the unwanted higher levels of proteases. PI genes have been characterized from various plant species (Habib and Fazili, 2007; Huang et al., 2007; Singh et al., 2009). By and large, PIs are primarily considered important as the prime defense against proteases released by pathogens (Habib and Fazili, 2007). However, PIs have also been implicated in various other physiological and developmental responses of plants (Jimenez et al., 2007; Wang et al., 2008). For instance, potential roles of PI have been suggested in seed germination, tapetum degeneration, programmed cell death etc. Wang et al. (2008) noted high level of OsPI8-1 promoter expression in shoot apical meristem and nodes on mature
* Corresponding author. E-mail address: [email protected] (A. Grover). http://dx.doi.org/10.1016/j.plaphy.2015.03.012 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.
culm along with little expression in nodes and internodes of young developing rice seedling. In addition, OsPI8-1 promoter showed inducibility in reproductive tissues like the middle layer and endothecium of anther and scutellar epithelium of germinating seeds. The expression of PI genes has been noted in salt, water deficit and temperature stress responses of plants (Dombrowski, 2003; Huang et al., 2007; Singh et al., 2009; Srinivasan et al., 2009). In peanut, the BowmaneBirk PI gene (BBI) was upregulated by water deficit and the level of transcript accumulation was higher in tolerant as compared to the susceptible cultivar (Drame et al., 2013). In chestnut, a cysteine-PI gene was induced by low temperature, salt and heat stresses (Pernas et al., 2000). Similarly a Kunitz-type PI was induced by heat and drought stress in Brassica (Satoh et al., 2001). Rice chymotrypsin PI (OCPI) gene family has 17 members (Singh et al., 2009). Huang et al. (2007) noted that rice OCPI1 gene is strongly induced by drought and salt stresses. In the latter study, it was further shown that the over-expression of OCPI1 gene in rice imparted enhanced drought resistance at the reproductive stages. Our group observed that OCPI2 gene (EMBL ID: AJ601438; TIGR ID:
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Os01g42860) along with OCPI1 gene shows induced transcript expression in response to salt, ABA, low temperature and mechanical injury (Singh et al., 2009). Furthermore, OCPI1 and OCPI2 genes were noted to be positioned in head-to-head orientation on chromosome number 2 of rice genome. Singh et al. (2009) showed that the inter-genic region between these two genes serves as a bidirectional promoter. Previous studies suggest that a bidirectional promoter is generally shared by two functionally-related genes (Kruglyak and Tang, 2000; Guan et al., 2004). Prompted by this argument and considering that OCPI1 and OCPI2 genes show coexpression profiling, this study aimed at investigating the functional roles of OCPI2 gene by reverse genetics approach. Arabidopsis transgenic plants produced with constitutive over-expression of OCPI2 gene showed enhanced growth under unstressed, control growth conditions and more vigorous vegetative and reproductive growth than the wild type (WT) plants. In addition, transgenic Arabidopsis plants over-expressing OCPI2 showed higher tolerance to salt and water stresses as against untransformed WT plants. 2. Material and methods 219 bp OCPI2 coding sequence was amplified from complementary DNA made from Pusa basmati 1 rice plants, using the requisite primers [forward primer-50 -CGGGATCCATGAGTTCATCAGACAGCAAG-30 ; reverse primer 50 -CGAGCTCGGGTACCTTAGCCAATCTTGGGAATC-30 ; extra sequence added for restriction enzyme digestions as shown by underline; Phusion™ high-fidelity polymerase (Finnzymes, Finland) used for PCR]. The PCR product was sub-cloned next to CaMV35S promoter in pBI121 vector. To produce transgenic Arabidopsis over-expressing OCPI2 (AtOCPI2) plants, Arabidopsis [Arabidopsis thaliana (L) Heynh ecotype Columbia (Col-0)] transformation was performed by floral dip method (Clough and Bent, 1998) using Agrobacterium tumefaciens strain AGL1. Phenotypic analyses were performed on T3 and T4 homozygous lines. For stress assays with seedlings, seeds of WT and AtOCPI2 plants were plated on ½ MS agar medium for germination in dark (2 d; 4 C) and then shifted to growth room (22 C, 16 h light/8 h dark cycle). 10-d-old AtOCPI2 and WT seedlings were subjected to mannitol (300 mM and 400 mM) and PEG6000 (2% and 3%) for osmotic and NaCl (100 mM, 200 mM) for salt stress treatments. For stress treatment with plants, 15-d-old AtOCPI2 and WT seedlings were transferred to pots for a week. For salt stress, plants were provided the requisite concentrations of NaCl (see Results). For detached leaf senescence assay, fully expanded leaves of 4-week old plants were placed on to 15 ml liquid ½ MS medium containing different concentrations of NaCl. For the quantitative-PCR (Q-PCR) analysis of OsCPI1 and OsCPI2 genes, total RNA was isolated using Trizol reagent from 10-d-old control and stressed (3% PEG for 6 h; 150 mM NaCl for 6 h) seedlings of rice (cultivar PB1). To analyze the expression level of selected marker genes in WT and AtOCPI2 plants, total RNA was isolated from 10-days-old,control and stressed (400 mM mannitol for 6 h; 200 mM NaCl for 4 h), seedlings using Trizol reagent. 2 ug of total RNA was reverse transcribed with a high capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative expression assays were performed with the SYBR green master mix kit and Stratagene Mx3005P sequence detection system according to the manufacturer's protocol (Agilent Technologies). Q-PCR conditions were as follows: 10 min at 95 C, 40 cycles of 30 s at 95 C, 30 s at 55 C, 30 s at 72 C. The relative quantification method (DDCT) was used to evaluate quantitative variation between replicates (three for each gene), and the actin gene was used as an internal control to normalize all data. List of primers used for assay of various stress marker genes is provided in Supplementary Table S1. Plant cell membrane stability indices (MSIs) were determined with
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20-d-old AtOCPI2 and WT seedlings using conductivity meter (CMD, UK) as per the standard method (Wang et al., 2011). Relative water contents (RWCs) were measured according to the standard methods (Wang et al., 2011). Proline content was measured spectrophotometrically (Bates et al., 1973). For the analysis of PI activity, 10-d-old AtOCPI2 and WT seedlings were transferred to ½ MS agar medium containing 200 mM NaCl (½ MS agar medium was used as control). Proteins were extracted in 50 mM Tris (pH 8.1) containing 20 mM CaCl2 (Xu et al., 2004). The PI activity against bovine chymotrypsin was determined by standard assays as described by Huang et al. (2007). In another assay, the bovine chymotrypsin was omitted to determine the endogenous chymotrypsin activities in WT and AtOCPI2 seedlings. For assaying the PI activity of purified OCPI2 protein, OCPI1 ORF was sub-cloned in pET-28a(þ) expression vector (Novagene) containing N-terminal His-coding sequence using gene specific primers (Supplementary Table S1). The construct was transformed in Escherichia coli (BL21 strain) cells. The recombinant protein containing N-terminal His-tag was purified by gel filtration using Sephacryl G200 (Supplementary Fig. S5). PI activity was assayed as described above using different concentrations of the purified OCPI2 protein. 3. Result In rice (cultivar PB1) seedlings, the expression of OsCPI2 transcript was higher as compared to OsCPI1 under both control and stressed (150 mM NaCl and 3% PEG) conditions (Supplementary Fig. S1). This prompted us to analyze the functional roles of OsCPI2 gene in greater depths. The construct designed with CaMV35S promoter driving OCPI2 gene, was transformed in Arabidopsis plants to yield AtOCPI2 progenies (Fig. 1A). T3 seeds of three lines i.e. AtOCPI2-1, AtOCPI2-2 and AtOCPI2-3 (transgenic nature confirmed by requisite PCRs) were selected for further analysis. Analysis with semi-quantitative PCR (sq-RT-PCR) showed significant constitutive expression of OCPI2 transcripts in the three AtOCPI2 lines (Supplementary Fig. S2). The vegetative growth of AtOCPI2 plants was noted to be more vigorous as compared to WT plants. Also, AtOCPI2 plants showed higher silique number, RWC values, proline content and membrane stability index (Fig. 1BeF; data presented for AtOCPI2-1 and AtOCPI23 lines while all three AtOCPI2 lines showed similar response in Fig. 1CeF). 10-d-old WT and AtOCPI2 seedlings were transferred to100 mM and 200 mM NaCl in ½ MS agar medium for the analysis of salt stress response. After 4 weeks of transfer to 100 mM NaCl, growth of both WT and AtOCPI2 plants was adversely affected. However, AtOCPI2 plants were affected to a much lesser extent than the WT plants in this treatment (Fig. 2A). WT seedlings almost died following 7 days of transfer to 200 mM NaCl. On the other hand, AtOCPI2 seedlings survived the stress (Supplementary Fig. S3A). Next, 4-weeks-old, soil grown AtOCPI2 and WT plants were subjected to 100 mM and 200 mM NaCl. After 2 weeks of NaCl treatment, WT plants withered while AtOCPI2 plants survived in the stress regime (Fig. 2B). The concentration of NaCl was further increased two fold at this point. Notably, AtOCPI2 plants were much less affected than the WT plants with the increased NaCl dosage. A significant difference in terms of cell membrane stability was observed for AtOCPI2 and WT plants after 20 days of salt stress: 200 mM NaCl treatment resulted in more injury to the cell membranes for WT plants as compared to the AtOCPI2 plants (Fig. 2C). In another set of experiments, detached WT and AtOCPI2 leaves were floated on 100 mM and 200 mM NaCl solutions. Treatment with NaCl resulted in a significantly accelerated rate of senescence in WT as compared to AtOCPI2 leaves (Supplementary Fig. S4). Next, 10-d-old AtOCPI2 seedlings were transferred to PEG (2%
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Fig. 1. (A) Diagrammatic representation of pBI121-OCPI2 construct employed for Arabidopsis transformation. (B) Growth of AtOCPI2 and WT plants under unstressed conditions. (C) Number of siliques/plant under unstressed conditions after four weeks growth in pot. (D) Relative water content (RWC) of AtOCPI2 and WT plants. (E) Proline content of AtOCPI2 and WT plants. (F) Membrane stability index of AtOCPI2 and WT plants.
and 3%) and mannitol (300 mM and 400 mM) for the analyses of osmotic stress response. After 20 days in 2% PEG, WT and AtOCPI2 plants appeared almost comparable. However, WT plants turned necrotic in appearance while AtOCPI2 plants appeared healthier at
3% PEG concentration in this duration (Supplementary Fig. S3B). After 30 days in 2% and 3% PEG, AtOCPI2 plants showed better adaptive growth than WT plants (Fig. 3A). Similar contrasting response was noted for WT and AtOCPI2 plants in response to
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Fig. 2. Salinity stress response of AtOCPI2 and WT plants. (A) Effect of 100 mM NaCl treatment. Plates were photographed 4 weeks after transfer of seedlings to stress medium. (B) Salt stress response of AtOCPI2 and WT plants in pots. Photographs of representative pots were taken two weeks (upper panel) and three weeks (lower panel) after stress treatment. The photographs are representative from three independent experiments. (C) Effect of 200 mM NaCl treatment on the membrane stability index of AtOCPI2 and WT plants.
mannitol treatment (300 mM, 400 mM) after 20 days (Supplementary Fig. S3C) and 30 days of stress (Fig. 3B). Next, PI enzyme activities were measured in unstressed and stressed plants. Crude proteins extracted from the AtOCPI2 and WT plants were assayed for PI activities with bovine chymotrypsin. The standard reaction with bovine chymotrypsin (DA256/min value) showed significantly lower CP activity than that of both stressed and unstressed AtOCPI2 and WT plants (Fig. 4A, B) suggesting the existence of endogenous chymotrypsin-like activities in crude
plant protein extracts. Notably, the increase of absorbance (DA256/ min) was less in the over-expression transgenic plants than that in the WT plants. Further, the endogenous chymotrypsin-like activities were determined in the AtOCPI2 plants using the same procedures as in the chymotrypsin inhibitory activity assay but with omission of bovine chymotrypsin from the reaction. The increase of absorbance (DA256/min) of the samples representing transgenic plants was significantly lower than the WT under both normal (Fig. 4C) and 200 mM NaCl stress (Fig. 4D) conditions. Further,
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Fig. 3. (A) Effect of PEG on the growth of seedlings. (B) Effect of mannitol on the growth of seedlings. Photographs were taken after 30 days from the transfer to stress medium.
when the purified OCPI2 protein was added, the absorbance values (DA256/min) were relatively lesser as compared to standard reaction (Fig. 4E). Transcript expression profiles of selected stress-related marker genes were analyzed using Q-PCR (details of the selected stressrelated genes shown in Supplementary Table S1). Out of the selected genes, the levels of alpha-amylase-like (AMY1), abscisic acid responsive element-binding factor 3 (ABF3), dehydration response element b1a (DREB1), pyrroline-5-carboxylate synthase (P5CS) and early response to desiccation 10 (ERD10) transcripts appeared higher in AtOCPI2 plants as compared to the WT plants under control conditions (Fig. 5A). However, transcript levels of proline dehydrogenase (ProDH) were higher in WT plants as compared to AtOCPI2 (Fig. 5A). Following mannitol treatment (400 mM, 6 h), the expression levels of CueZn super oxide dismutase (CueZn SOD) transcripts appeared higher in AtOCPI2 plants in comparison with the WT plants (Fig. 5B). Level of ProDH transcripts was higher in WT plants compared to AtOCPI2 plants in mannitol stress also (Fig. 5B). Following NaCl stress (200 mM, 4 h), expression levels of AMY1 and DREB1 transcripts appeared higher
with a lower ProDH transcript levels in AtOCPI2 plants than the WT plants (Fig. 5C). 4. Discussion Plant PI genes have been implicated in a wide range of plant processes including growth and development as well as response to biotic and abiotic stresses. Out of the 17 known members in rice CPI gene family, OCPI1 and OCPI2 genes share a common, bidirectional promoter and are co-expressed with strong induction under salt and osmotic stress (Singh et al., 2009). In the previous work, over-expression of OCPI1 in rice was correlated with enhancement in drought tolerance during reproductive stages (Huang et al., 2007). From the transcript expression analysis, it accrued that OsCPI2 levels is several folds higher than OsCPI1 under stressed as well as unstressed, control conditions (Supplementary Fig. S1). Prompted by this observation, we raised homozygous Arabidopsis transgenic to analyze how over-expression of OCPI2 would affect the phenotype of the plants. Importantly, AtOCPI2 plants showed enhanced vegetative and reproductive potential in contrast to WT
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Fig. 4. Chymotrypsin activity assays in protein extracts of Arabidopsis seedlings. Chymotrypsin assays with bovine chymotrypsin in unstressed control (A) and 200 mM salt stressed plants (B). Chymotrypsin activity assays without bovine chymotrypsin in unstressed control (C) and 200 mM salt stressed plants (D). Chymotrypsin activity assays using 65 ug purified protein (E).
plants under normal growing conditions. There was no phenotypic aberrations and yield penalty as a result of the over-expression of this gene. The AtOCPI2 plants were phenotypically more robust with higher biomass and productive (more number of siliques) resulting into an increased seed yield in comparison to WT plants. Our findings with positive effects of the OCPI2 over-expression in Arabidopsis as trans-host under control conditions are in contrast with the earlier work by Huang et al. (2007) where over-expression
of OCPI1 in rice did not show any enhancement in growth and potential yield under normal growth conditions. Apart from exhibiting growth advantage, AtOCPI2 plants were more tolerant towards salt stress. AtOCPI2 plants exhibited better growth than WT plants at lower salt concentration (100 mM NaCl). The AtOCPI2 plants survived higher salt concentrations (200 mM) unlike WT seedlings that withered during the first week. The mature, soilgrown AtOCPI2 plants also exhibited higher salt tolerance as
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Fig. 5. Quantitative RT-PCR for stress marker genes in AtOCPI2 and WT plants. (A) Expression profiles of stress marker genes in unstressed conditions. (B) Expression profiles of stress marker genes in mannitol stress. (C) Expression profiles of stress marker genes in NaCl stress. Y axis represents the fold change.
against WT (Fig. 2B). Similarly, higher salt tolerance of AtOCPI2 leaves was noted under induced senescence conditions (Supplementary Fig. S4). Detachment of leaves triggers wounding response resulting into an increase in over-all proteinases content (Miller and Huffaker, 1985; Roberts et al., 2012). Stress-induced senescence and chlorophyll leaching in detached leaves of AtOCPI2 plants in contrast to WT were delayed advocating the positive effects of OCPI2 over-expression against the enhanced endogenous proteases levels of the system. As noted for salt stress, AtOCPI2 plants were more tolerant to osmotic stress also. AtOCPI2
seedlings maintained much healthier growth than the WT seedlings in response to PEG and mannitol treatments. The results of the physiological and biochemical parameters tested in this study corroborate the phenotype of the transgenic plants. The higher values for membrane stability index, RWC and proline content of AtOCPI2 plants appear as the factors attributing to the enhanced growth and stress tolerant phenotypes. The higher water retention capacity could be the reason for higher biomass and better growth of the transgenic plants. In addition, the higher membrane stability index of transgenic plants resulted in lesser membrane injury as compared to the WT both in stressed and unstressed conditions. The selected genes which show characteristic altered expression during NaCl and osmotic stresses in plants are often regarded as marker genes (Wang et al., 2011). The enhanced growth of AtOCPI2 plants under unstressed control, conditions as well as increased tolerance to salt and osmotic stresses was corroborated by expression profiles of selected stress-related markers (Supplementary Table S1). The transcript levels of AMY1, ABF3, DREB1, P5CS and ERD10 were higher in AtOCPI2 than WT plants under unstressed control conditions. The increased expression of these genes may account for the vigorous growth of AtOCPI2 plants under control conditions. Further, levels of CueZn SOD in case of mannitol treatments and levels of AMY1, DREB1 in case of NaCl treatments were higher in AtOCPI2 plants than WT plants, possibly supporting the survival of transgenic under these stresses. These high transcript levels may be contributing towards higher growth phenotype observed for AtOCPI2 plants in this study. The higher level of P5CS along with lower ProDH transcript levels in AtOCPI2 plants possibly accounts for their higher proline levels noted in this study. Proline has a role in osmotic adjustments, protection of macromolecules during dehydration and it also function as a hydroxyl radial scavenger (Szabados and Savoure, 2010). Increasing the titer of proline by altering the proline biosynthetic enzyme levels have been shown to increase the salt and drought tolerance of transgenic plants (Liang et al., 2013). Increased level of proline in AtOCPI2 plants is an added advantage towards its enhanced tolerance against salt and osmotic stresses. We further noted that chymotrypsin protease activity was lower in AtOCPI2 plants as compared to WT under both unstressed and stressed conditions. Also, the suppression of chymotrypsin activity by purified OCPI2 (indicated by the low absorbance value obtained in Fig. 4E) suggested PI activity in this protein. Based on these observations, we speculate that increased levels of OCPI2 protein contribute to decreased chymotrypsin protease activity of AtOCPI2 plants. We further argue that decreased chymotrypsin protease activities may be the basis for the higher biomass and abiotic stress tolerance phenotype of the AtOCPI2 as compared to WT plants. Huang et al. (2007) correlated the enhanced drought tolerance of OCPI1 over-expressing rice plants with the stronger proteaseinhibitory activity in their protein extracts along with less decrease of total proteins than the WT under drought stress. Therefore, the decreased activities of proteases may be a critical factor in enhancing stress tolerance noted with over-expression of OCPI2 in Aabidopsis. The marked differences regarding the growth and yield enhancement under normal growth conditions with OCPI2 over-expression in Arabidopsis (this study) over OCPI1 overexpression in rice by Huang et al. (2007) highlights the possible differences in the mode of action and selection of target proteases by the two inhibitors. Furthermore, the efficacy of OCPI2 overexpression over OCPI1 is also advocated. It was interesting to note that there are six Arabidopsis serine protease inhibitor gene accessions (AT5G43580, AT3G46860, AT5G43570, AT2G38900, AT3G50020 and AT2G38870) orthologous to OCPI1/OCPI2 genes. Of these six genes, AT5G43580 was inducible in response to osmotic stress condition as per the Genevestigator (https://genevestigator.
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com/) data. These proteins inhibit different target proteases as suggested by MEROPS database (http://merops.sanger.ac.uk; Supplementary Table S2). In future studies, it would be interesting to explore the precise targets of these inhibitors in plants. Salt stress affects ~7% of the total land area in the world (Tran and Mochida, 2010). Salt stress results in both ionic and osmotic stresses, which further lead to secondary stresses such as oxidative stress and nutrient deficiency stress (Chinnusamy et al., 2006; Sahi et al., 2006). It negatively affects photosynthesis, energy production, lipid metabolism, integrity of membranes and enzyme activities (Chinnusamy et al., 2006; Sahi et al., 2006). By over-expressing a tobacco PI, Srinivasan et al. (2009) generated transgenic tobacco plants exhibiting enhanced tolerance to salt (NaCl) and sorbitol. This group also noted that the transgenic plants show enhanced seed germination, root length and root-shoot ratio, significantly enhanced total chlorophyll content and reduced thiobarbituric acid-reactive substances under stress. It thus appears that besides their role in biotic stresses, PI genes certainly have roles in abiotic stresses across the spectrum of plant species. We propose that OCPI1 and OCPI2 (a divergent gene pair with ability to enhance growth and yield under unstressed conditions [OCPI2] and impart stress tolerance [OCPI1, OCPI2]) provide us an interesting locus that can be used further in marker-assisted breeding. Conversely, the two genes can be used together as a cassette in transgenic approach to enhance the genetic pool as well as to engineer stress tolerance in rice. Considering the advantages of PI proteins over-expression in terms of enhancement in tolerance against biotic stresses, it should be important to address the biotic and abiotic stress phenotype together with the paired OCPI1 and OCPI2 genes in future studies. Acknowledgments LDT acknowledges the University Grants Commission (UGC) for the fellowship award. DM and RM acknowledge Council of Scientific and Industrial Research (CSIR), New Delhi for the fellowship awards. AG is thankful to the Department of Science and Technology, Government of India, for the JC Bose National Fellowship award. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2015.03.012. Contribution LDT, DM and RCM planned and executed the experiments on cloning of OCPI2 gene, transformation of Arabidopsis and phenotype analysis. AG participated in planning, data analysis and writing of the manuscript.
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Contents lists available at ScienceDirect
Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants Lalit Dev Tiwari, Dheeraj Mittal, Ratnesh Chandra Mishra, Anil Grover* Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 February 2015 Accepted 31 March 2015 Available online 16 April 2015
Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Arabidopsis Chymotrypsin protease inhibitor Growth Osmotic stress Rice Salt stress
1. Introduction Along with proteases and their substrate proteins, protease inhibitors (PI) constitute an important component of the protein turnover machinery. PIs keep a check on the otherwise damaging effects of the unwanted higher levels of proteases. PI genes have been characterized from various plant species (Habib and Fazili, 2007; Huang et al., 2007; Singh et al., 2009). By and large, PIs are primarily considered important as the prime defense against proteases released by pathogens (Habib and Fazili, 2007). However, PIs have also been implicated in various other physiological and developmental responses of plants (Jimenez et al., 2007; Wang et al., 2008). For instance, potential roles of PI have been suggested in seed germination, tapetum degeneration, programmed cell death etc. Wang et al. (2008) noted high level of OsPI8-1 promoter expression in shoot apical meristem and nodes on mature
* Corresponding author. E-mail address: [email protected] (A. Grover). http://dx.doi.org/10.1016/j.plaphy.2015.03.012 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.
culm along with little expression in nodes and internodes of young developing rice seedling. In addition, OsPI8-1 promoter showed inducibility in reproductive tissues like the middle layer and endothecium of anther and scutellar epithelium of germinating seeds. The expression of PI genes has been noted in salt, water deficit and temperature stress responses of plants (Dombrowski, 2003; Huang et al., 2007; Singh et al., 2009; Srinivasan et al., 2009). In peanut, the BowmaneBirk PI gene (BBI) was upregulated by water deficit and the level of transcript accumulation was higher in tolerant as compared to the susceptible cultivar (Drame et al., 2013). In chestnut, a cysteine-PI gene was induced by low temperature, salt and heat stresses (Pernas et al., 2000). Similarly a Kunitz-type PI was induced by heat and drought stress in Brassica (Satoh et al., 2001). Rice chymotrypsin PI (OCPI) gene family has 17 members (Singh et al., 2009). Huang et al. (2007) noted that rice OCPI1 gene is strongly induced by drought and salt stresses. In the latter study, it was further shown that the over-expression of OCPI1 gene in rice imparted enhanced drought resistance at the reproductive stages. Our group observed that OCPI2 gene (EMBL ID: AJ601438; TIGR ID:
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Os01g42860) along with OCPI1 gene shows induced transcript expression in response to salt, ABA, low temperature and mechanical injury (Singh et al., 2009). Furthermore, OCPI1 and OCPI2 genes were noted to be positioned in head-to-head orientation on chromosome number 2 of rice genome. Singh et al. (2009) showed that the inter-genic region between these two genes serves as a bidirectional promoter. Previous studies suggest that a bidirectional promoter is generally shared by two functionally-related genes (Kruglyak and Tang, 2000; Guan et al., 2004). Prompted by this argument and considering that OCPI1 and OCPI2 genes show coexpression profiling, this study aimed at investigating the functional roles of OCPI2 gene by reverse genetics approach. Arabidopsis transgenic plants produced with constitutive over-expression of OCPI2 gene showed enhanced growth under unstressed, control growth conditions and more vigorous vegetative and reproductive growth than the wild type (WT) plants. In addition, transgenic Arabidopsis plants over-expressing OCPI2 showed higher tolerance to salt and water stresses as against untransformed WT plants. 2. Material and methods 219 bp OCPI2 coding sequence was amplified from complementary DNA made from Pusa basmati 1 rice plants, using the requisite primers [forward primer-50 -CGGGATCCATGAGTTCATCAGACAGCAAG-30 ; reverse primer 50 -CGAGCTCGGGTACCTTAGCCAATCTTGGGAATC-30 ; extra sequence added for restriction enzyme digestions as shown by underline; Phusion™ high-fidelity polymerase (Finnzymes, Finland) used for PCR]. The PCR product was sub-cloned next to CaMV35S promoter in pBI121 vector. To produce transgenic Arabidopsis over-expressing OCPI2 (AtOCPI2) plants, Arabidopsis [Arabidopsis thaliana (L) Heynh ecotype Columbia (Col-0)] transformation was performed by floral dip method (Clough and Bent, 1998) using Agrobacterium tumefaciens strain AGL1. Phenotypic analyses were performed on T3 and T4 homozygous lines. For stress assays with seedlings, seeds of WT and AtOCPI2 plants were plated on ½ MS agar medium for germination in dark (2 d; 4 C) and then shifted to growth room (22 C, 16 h light/8 h dark cycle). 10-d-old AtOCPI2 and WT seedlings were subjected to mannitol (300 mM and 400 mM) and PEG6000 (2% and 3%) for osmotic and NaCl (100 mM, 200 mM) for salt stress treatments. For stress treatment with plants, 15-d-old AtOCPI2 and WT seedlings were transferred to pots for a week. For salt stress, plants were provided the requisite concentrations of NaCl (see Results). For detached leaf senescence assay, fully expanded leaves of 4-week old plants were placed on to 15 ml liquid ½ MS medium containing different concentrations of NaCl. For the quantitative-PCR (Q-PCR) analysis of OsCPI1 and OsCPI2 genes, total RNA was isolated using Trizol reagent from 10-d-old control and stressed (3% PEG for 6 h; 150 mM NaCl for 6 h) seedlings of rice (cultivar PB1). To analyze the expression level of selected marker genes in WT and AtOCPI2 plants, total RNA was isolated from 10-days-old,control and stressed (400 mM mannitol for 6 h; 200 mM NaCl for 4 h), seedlings using Trizol reagent. 2 ug of total RNA was reverse transcribed with a high capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative expression assays were performed with the SYBR green master mix kit and Stratagene Mx3005P sequence detection system according to the manufacturer's protocol (Agilent Technologies). Q-PCR conditions were as follows: 10 min at 95 C, 40 cycles of 30 s at 95 C, 30 s at 55 C, 30 s at 72 C. The relative quantification method (DDCT) was used to evaluate quantitative variation between replicates (three for each gene), and the actin gene was used as an internal control to normalize all data. List of primers used for assay of various stress marker genes is provided in Supplementary Table S1. Plant cell membrane stability indices (MSIs) were determined with
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20-d-old AtOCPI2 and WT seedlings using conductivity meter (CMD, UK) as per the standard method (Wang et al., 2011). Relative water contents (RWCs) were measured according to the standard methods (Wang et al., 2011). Proline content was measured spectrophotometrically (Bates et al., 1973). For the analysis of PI activity, 10-d-old AtOCPI2 and WT seedlings were transferred to ½ MS agar medium containing 200 mM NaCl (½ MS agar medium was used as control). Proteins were extracted in 50 mM Tris (pH 8.1) containing 20 mM CaCl2 (Xu et al., 2004). The PI activity against bovine chymotrypsin was determined by standard assays as described by Huang et al. (2007). In another assay, the bovine chymotrypsin was omitted to determine the endogenous chymotrypsin activities in WT and AtOCPI2 seedlings. For assaying the PI activity of purified OCPI2 protein, OCPI1 ORF was sub-cloned in pET-28a(þ) expression vector (Novagene) containing N-terminal His-coding sequence using gene specific primers (Supplementary Table S1). The construct was transformed in Escherichia coli (BL21 strain) cells. The recombinant protein containing N-terminal His-tag was purified by gel filtration using Sephacryl G200 (Supplementary Fig. S5). PI activity was assayed as described above using different concentrations of the purified OCPI2 protein. 3. Result In rice (cultivar PB1) seedlings, the expression of OsCPI2 transcript was higher as compared to OsCPI1 under both control and stressed (150 mM NaCl and 3% PEG) conditions (Supplementary Fig. S1). This prompted us to analyze the functional roles of OsCPI2 gene in greater depths. The construct designed with CaMV35S promoter driving OCPI2 gene, was transformed in Arabidopsis plants to yield AtOCPI2 progenies (Fig. 1A). T3 seeds of three lines i.e. AtOCPI2-1, AtOCPI2-2 and AtOCPI2-3 (transgenic nature confirmed by requisite PCRs) were selected for further analysis. Analysis with semi-quantitative PCR (sq-RT-PCR) showed significant constitutive expression of OCPI2 transcripts in the three AtOCPI2 lines (Supplementary Fig. S2). The vegetative growth of AtOCPI2 plants was noted to be more vigorous as compared to WT plants. Also, AtOCPI2 plants showed higher silique number, RWC values, proline content and membrane stability index (Fig. 1BeF; data presented for AtOCPI2-1 and AtOCPI23 lines while all three AtOCPI2 lines showed similar response in Fig. 1CeF). 10-d-old WT and AtOCPI2 seedlings were transferred to100 mM and 200 mM NaCl in ½ MS agar medium for the analysis of salt stress response. After 4 weeks of transfer to 100 mM NaCl, growth of both WT and AtOCPI2 plants was adversely affected. However, AtOCPI2 plants were affected to a much lesser extent than the WT plants in this treatment (Fig. 2A). WT seedlings almost died following 7 days of transfer to 200 mM NaCl. On the other hand, AtOCPI2 seedlings survived the stress (Supplementary Fig. S3A). Next, 4-weeks-old, soil grown AtOCPI2 and WT plants were subjected to 100 mM and 200 mM NaCl. After 2 weeks of NaCl treatment, WT plants withered while AtOCPI2 plants survived in the stress regime (Fig. 2B). The concentration of NaCl was further increased two fold at this point. Notably, AtOCPI2 plants were much less affected than the WT plants with the increased NaCl dosage. A significant difference in terms of cell membrane stability was observed for AtOCPI2 and WT plants after 20 days of salt stress: 200 mM NaCl treatment resulted in more injury to the cell membranes for WT plants as compared to the AtOCPI2 plants (Fig. 2C). In another set of experiments, detached WT and AtOCPI2 leaves were floated on 100 mM and 200 mM NaCl solutions. Treatment with NaCl resulted in a significantly accelerated rate of senescence in WT as compared to AtOCPI2 leaves (Supplementary Fig. S4). Next, 10-d-old AtOCPI2 seedlings were transferred to PEG (2%
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Fig. 1. (A) Diagrammatic representation of pBI121-OCPI2 construct employed for Arabidopsis transformation. (B) Growth of AtOCPI2 and WT plants under unstressed conditions. (C) Number of siliques/plant under unstressed conditions after four weeks growth in pot. (D) Relative water content (RWC) of AtOCPI2 and WT plants. (E) Proline content of AtOCPI2 and WT plants. (F) Membrane stability index of AtOCPI2 and WT plants.
and 3%) and mannitol (300 mM and 400 mM) for the analyses of osmotic stress response. After 20 days in 2% PEG, WT and AtOCPI2 plants appeared almost comparable. However, WT plants turned necrotic in appearance while AtOCPI2 plants appeared healthier at
3% PEG concentration in this duration (Supplementary Fig. S3B). After 30 days in 2% and 3% PEG, AtOCPI2 plants showed better adaptive growth than WT plants (Fig. 3A). Similar contrasting response was noted for WT and AtOCPI2 plants in response to
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Fig. 2. Salinity stress response of AtOCPI2 and WT plants. (A) Effect of 100 mM NaCl treatment. Plates were photographed 4 weeks after transfer of seedlings to stress medium. (B) Salt stress response of AtOCPI2 and WT plants in pots. Photographs of representative pots were taken two weeks (upper panel) and three weeks (lower panel) after stress treatment. The photographs are representative from three independent experiments. (C) Effect of 200 mM NaCl treatment on the membrane stability index of AtOCPI2 and WT plants.
mannitol treatment (300 mM, 400 mM) after 20 days (Supplementary Fig. S3C) and 30 days of stress (Fig. 3B). Next, PI enzyme activities were measured in unstressed and stressed plants. Crude proteins extracted from the AtOCPI2 and WT plants were assayed for PI activities with bovine chymotrypsin. The standard reaction with bovine chymotrypsin (DA256/min value) showed significantly lower CP activity than that of both stressed and unstressed AtOCPI2 and WT plants (Fig. 4A, B) suggesting the existence of endogenous chymotrypsin-like activities in crude
plant protein extracts. Notably, the increase of absorbance (DA256/ min) was less in the over-expression transgenic plants than that in the WT plants. Further, the endogenous chymotrypsin-like activities were determined in the AtOCPI2 plants using the same procedures as in the chymotrypsin inhibitory activity assay but with omission of bovine chymotrypsin from the reaction. The increase of absorbance (DA256/min) of the samples representing transgenic plants was significantly lower than the WT under both normal (Fig. 4C) and 200 mM NaCl stress (Fig. 4D) conditions. Further,
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Fig. 3. (A) Effect of PEG on the growth of seedlings. (B) Effect of mannitol on the growth of seedlings. Photographs were taken after 30 days from the transfer to stress medium.
when the purified OCPI2 protein was added, the absorbance values (DA256/min) were relatively lesser as compared to standard reaction (Fig. 4E). Transcript expression profiles of selected stress-related marker genes were analyzed using Q-PCR (details of the selected stressrelated genes shown in Supplementary Table S1). Out of the selected genes, the levels of alpha-amylase-like (AMY1), abscisic acid responsive element-binding factor 3 (ABF3), dehydration response element b1a (DREB1), pyrroline-5-carboxylate synthase (P5CS) and early response to desiccation 10 (ERD10) transcripts appeared higher in AtOCPI2 plants as compared to the WT plants under control conditions (Fig. 5A). However, transcript levels of proline dehydrogenase (ProDH) were higher in WT plants as compared to AtOCPI2 (Fig. 5A). Following mannitol treatment (400 mM, 6 h), the expression levels of CueZn super oxide dismutase (CueZn SOD) transcripts appeared higher in AtOCPI2 plants in comparison with the WT plants (Fig. 5B). Level of ProDH transcripts was higher in WT plants compared to AtOCPI2 plants in mannitol stress also (Fig. 5B). Following NaCl stress (200 mM, 4 h), expression levels of AMY1 and DREB1 transcripts appeared higher
with a lower ProDH transcript levels in AtOCPI2 plants than the WT plants (Fig. 5C). 4. Discussion Plant PI genes have been implicated in a wide range of plant processes including growth and development as well as response to biotic and abiotic stresses. Out of the 17 known members in rice CPI gene family, OCPI1 and OCPI2 genes share a common, bidirectional promoter and are co-expressed with strong induction under salt and osmotic stress (Singh et al., 2009). In the previous work, over-expression of OCPI1 in rice was correlated with enhancement in drought tolerance during reproductive stages (Huang et al., 2007). From the transcript expression analysis, it accrued that OsCPI2 levels is several folds higher than OsCPI1 under stressed as well as unstressed, control conditions (Supplementary Fig. S1). Prompted by this observation, we raised homozygous Arabidopsis transgenic to analyze how over-expression of OCPI2 would affect the phenotype of the plants. Importantly, AtOCPI2 plants showed enhanced vegetative and reproductive potential in contrast to WT
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Fig. 4. Chymotrypsin activity assays in protein extracts of Arabidopsis seedlings. Chymotrypsin assays with bovine chymotrypsin in unstressed control (A) and 200 mM salt stressed plants (B). Chymotrypsin activity assays without bovine chymotrypsin in unstressed control (C) and 200 mM salt stressed plants (D). Chymotrypsin activity assays using 65 ug purified protein (E).
plants under normal growing conditions. There was no phenotypic aberrations and yield penalty as a result of the over-expression of this gene. The AtOCPI2 plants were phenotypically more robust with higher biomass and productive (more number of siliques) resulting into an increased seed yield in comparison to WT plants. Our findings with positive effects of the OCPI2 over-expression in Arabidopsis as trans-host under control conditions are in contrast with the earlier work by Huang et al. (2007) where over-expression
of OCPI1 in rice did not show any enhancement in growth and potential yield under normal growth conditions. Apart from exhibiting growth advantage, AtOCPI2 plants were more tolerant towards salt stress. AtOCPI2 plants exhibited better growth than WT plants at lower salt concentration (100 mM NaCl). The AtOCPI2 plants survived higher salt concentrations (200 mM) unlike WT seedlings that withered during the first week. The mature, soilgrown AtOCPI2 plants also exhibited higher salt tolerance as
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Fig. 5. Quantitative RT-PCR for stress marker genes in AtOCPI2 and WT plants. (A) Expression profiles of stress marker genes in unstressed conditions. (B) Expression profiles of stress marker genes in mannitol stress. (C) Expression profiles of stress marker genes in NaCl stress. Y axis represents the fold change.
against WT (Fig. 2B). Similarly, higher salt tolerance of AtOCPI2 leaves was noted under induced senescence conditions (Supplementary Fig. S4). Detachment of leaves triggers wounding response resulting into an increase in over-all proteinases content (Miller and Huffaker, 1985; Roberts et al., 2012). Stress-induced senescence and chlorophyll leaching in detached leaves of AtOCPI2 plants in contrast to WT were delayed advocating the positive effects of OCPI2 over-expression against the enhanced endogenous proteases levels of the system. As noted for salt stress, AtOCPI2 plants were more tolerant to osmotic stress also. AtOCPI2
seedlings maintained much healthier growth than the WT seedlings in response to PEG and mannitol treatments. The results of the physiological and biochemical parameters tested in this study corroborate the phenotype of the transgenic plants. The higher values for membrane stability index, RWC and proline content of AtOCPI2 plants appear as the factors attributing to the enhanced growth and stress tolerant phenotypes. The higher water retention capacity could be the reason for higher biomass and better growth of the transgenic plants. In addition, the higher membrane stability index of transgenic plants resulted in lesser membrane injury as compared to the WT both in stressed and unstressed conditions. The selected genes which show characteristic altered expression during NaCl and osmotic stresses in plants are often regarded as marker genes (Wang et al., 2011). The enhanced growth of AtOCPI2 plants under unstressed control, conditions as well as increased tolerance to salt and osmotic stresses was corroborated by expression profiles of selected stress-related markers (Supplementary Table S1). The transcript levels of AMY1, ABF3, DREB1, P5CS and ERD10 were higher in AtOCPI2 than WT plants under unstressed control conditions. The increased expression of these genes may account for the vigorous growth of AtOCPI2 plants under control conditions. Further, levels of CueZn SOD in case of mannitol treatments and levels of AMY1, DREB1 in case of NaCl treatments were higher in AtOCPI2 plants than WT plants, possibly supporting the survival of transgenic under these stresses. These high transcript levels may be contributing towards higher growth phenotype observed for AtOCPI2 plants in this study. The higher level of P5CS along with lower ProDH transcript levels in AtOCPI2 plants possibly accounts for their higher proline levels noted in this study. Proline has a role in osmotic adjustments, protection of macromolecules during dehydration and it also function as a hydroxyl radial scavenger (Szabados and Savoure, 2010). Increasing the titer of proline by altering the proline biosynthetic enzyme levels have been shown to increase the salt and drought tolerance of transgenic plants (Liang et al., 2013). Increased level of proline in AtOCPI2 plants is an added advantage towards its enhanced tolerance against salt and osmotic stresses. We further noted that chymotrypsin protease activity was lower in AtOCPI2 plants as compared to WT under both unstressed and stressed conditions. Also, the suppression of chymotrypsin activity by purified OCPI2 (indicated by the low absorbance value obtained in Fig. 4E) suggested PI activity in this protein. Based on these observations, we speculate that increased levels of OCPI2 protein contribute to decreased chymotrypsin protease activity of AtOCPI2 plants. We further argue that decreased chymotrypsin protease activities may be the basis for the higher biomass and abiotic stress tolerance phenotype of the AtOCPI2 as compared to WT plants. Huang et al. (2007) correlated the enhanced drought tolerance of OCPI1 over-expressing rice plants with the stronger proteaseinhibitory activity in their protein extracts along with less decrease of total proteins than the WT under drought stress. Therefore, the decreased activities of proteases may be a critical factor in enhancing stress tolerance noted with over-expression of OCPI2 in Aabidopsis. The marked differences regarding the growth and yield enhancement under normal growth conditions with OCPI2 over-expression in Arabidopsis (this study) over OCPI1 overexpression in rice by Huang et al. (2007) highlights the possible differences in the mode of action and selection of target proteases by the two inhibitors. Furthermore, the efficacy of OCPI2 overexpression over OCPI1 is also advocated. It was interesting to note that there are six Arabidopsis serine protease inhibitor gene accessions (AT5G43580, AT3G46860, AT5G43570, AT2G38900, AT3G50020 and AT2G38870) orthologous to OCPI1/OCPI2 genes. Of these six genes, AT5G43580 was inducible in response to osmotic stress condition as per the Genevestigator (https://genevestigator.
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com/) data. These proteins inhibit different target proteases as suggested by MEROPS database (http://merops.sanger.ac.uk; Supplementary Table S2). In future studies, it would be interesting to explore the precise targets of these inhibitors in plants. Salt stress affects ~7% of the total land area in the world (Tran and Mochida, 2010). Salt stress results in both ionic and osmotic stresses, which further lead to secondary stresses such as oxidative stress and nutrient deficiency stress (Chinnusamy et al., 2006; Sahi et al., 2006). It negatively affects photosynthesis, energy production, lipid metabolism, integrity of membranes and enzyme activities (Chinnusamy et al., 2006; Sahi et al., 2006). By over-expressing a tobacco PI, Srinivasan et al. (2009) generated transgenic tobacco plants exhibiting enhanced tolerance to salt (NaCl) and sorbitol. This group also noted that the transgenic plants show enhanced seed germination, root length and root-shoot ratio, significantly enhanced total chlorophyll content and reduced thiobarbituric acid-reactive substances under stress. It thus appears that besides their role in biotic stresses, PI genes certainly have roles in abiotic stresses across the spectrum of plant species. We propose that OCPI1 and OCPI2 (a divergent gene pair with ability to enhance growth and yield under unstressed conditions [OCPI2] and impart stress tolerance [OCPI1, OCPI2]) provide us an interesting locus that can be used further in marker-assisted breeding. Conversely, the two genes can be used together as a cassette in transgenic approach to enhance the genetic pool as well as to engineer stress tolerance in rice. Considering the advantages of PI proteins over-expression in terms of enhancement in tolerance against biotic stresses, it should be important to address the biotic and abiotic stress phenotype together with the paired OCPI1 and OCPI2 genes in future studies. Acknowledgments LDT acknowledges the University Grants Commission (UGC) for the fellowship award. DM and RM acknowledge Council of Scientific and Industrial Research (CSIR), New Delhi for the fellowship awards. AG is thankful to the Department of Science and Technology, Government of India, for the JC Bose National Fellowship award. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2015.03.012. Contribution LDT, DM and RCM planned and executed the experiments on cloning of OCPI2 gene, transformation of Arabidopsis and phenotype analysis. AG participated in planning, data analysis and writing of the manuscript.
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