Effect of carbonic anhydrase-containing endophytic bacteria on growth and physiological attributes of wheat under water-deficit conditions

ABSTRACT Drought is one of the major limitations to agricultural productivity, suppressing plant growth and yield of food crops throughout the world particularly in arid and semiarid regions. Drought-tolerant carbonic anhydrase (CA; EC 4.2.1.1)-containing endophytic bacteria may improve plant growth under stressed conditions. In the present study, effect of drought-tolerant CA-containing endophytic bacteria on growth and physiology of wheat under water-deficit conditions was studied. One hundred and fifty isolates were isolated from wheat plants and screened for their ability to tolerate polyethylene glycol (PEG) 6000-induced water-deficit stress (−0.31 to −3.20 MPa). Fifty isolates exhibiting intrinsic ability to tolerate stress were further screened for CA activity. Ten drought-tolerant isolates with higher CA activity were evaluated for improving wheat growth under water-deficit conditions (−0.04, −1.09, −1.23 MPa). Results showed that PEG-mediated water-deficit stress significantly reduced growth of wheat. However, inoculation with isolates WR2, WS11 and WL19 significantly enhanced seedling growth by improving maximum root length, shoot length, root and shoot dry weight under non-stressed as well as stressed conditions. These isolates were identified by 16S rRNA as Bacillus marisflavi (WR2) Bacillus thuringiensis (WS11) and Bacillus subtilis (WL19). Isolate WL19 also improved chlorophyll content, photosynthetic rate, CA activity and relative water content compared to uninoculated control plants. Overall, our findings suggest that endophytic bacterial isolates WR2, WS11 and WL19 with CA activity can enhance photosynthesis and biomass of wheat seedlings under water-deficit conditions. Abbreviations: CA: Carbonic anhydrase; PEG: Polyethylene glycol; CO2: Carbon dioxide; HCO3–: Bicarbonate; TSA: Tryptic Soy Agar; LB: Luria Bertani; A: CO2 assimilation rate; E: Transpiration rate; gs: Stomatal conductance; Ci: Substomatal CO2 concentration; RWC: Relative water content; EL: Electrolyte leakage


Introduction
Climate change is a serious threat to productivity of food crops throughout the world due to rise in temperature and erratic change in rainfall pattern. The global temperature is predicted to increase drastically from 1.8 to 3.6°C by the year 2100 (Sharma, Bhattacharyyal, Rajkhowa, & Jha, 2014). Such climatic variables may project to increasingly hotter summer and severe drought stress in arid and semiarid regions. The drought area is predicted to increase 2-fold while water resources will decline up to 30% by the year 2050 (Falkenmark, 2013). Plants growing under water-deficit conditions face limited water supply and nutrient deficiencies. In agricultural regions, up to 50% or more yield loss can be experienced by drought (Wood, 2005). Drought impairs plant growth and development by reducing leaf size, stem thickness, root proliferation and disturbing plant water relations. It induces a range of changes in physiological and cellular functions of plants such as reduced carbon dioxide assimilation, membrane damage and disturbed enzymatic activity (Farooq, Wahid, Kobayashi, Fujita, & Basra, 2009). Reduced stomatal aperture and photosynthetic rate under severe water-deficit conditions thereby decrease crop yields (Anjum et al., 2011). Therefore, increasing productivity per drop of water is becoming important for many regions.
Several measures have been adopted to mitigate the drought-induced yield reduction and produce high and satisfactory yield under stress environments. Molecular breeding programs and transgenic approaches have been used to develop drought-tolerant plants but these have certain limitations (Cominelli, Sala, Calvi, Gusmaroli, & Tonelli, 2008;Cominelli, Conti, Tonelli, & Galbiati, 2013). Plant growth-promoting rhizobacteria (PGPR) have also been reported to mitigate the adverse effects of drought (Vardharajula, Ali, Grover, Reddy, & Bandi, 2011). However, survival of PGPR may not be sustainable for longer run due to rhizospheric competition and various environmental injuries like drought, salinity, etc. Recently, inoculation of plants with endophytic bacteria to enhance the drought tolerance (Akbari, Akbari, & Golakiya, 2016;Khan et al., 2016) has gained interest among the scientific community. Compared to other bacteria which colonize the plant epiphytically, endophytes are better protected from the environmental stresses like temperature, osmotic potential and ultraviolet radiation. Endophytic bacteria colonize the internal plant tissues without causing any negative effect and sign of infection to the host plant (Schulz & Boyle, 2006). These bacteria are almost present in all plants. They reside inside the plant in intercellular, in vascular bundle and within the cell (Ulrich, Ulrich, & Ewald, 2008). They can promote plant growth and development by producing variety of products that could be beneficial to the host plant (Ryan, Germaine, Franks, Ryan, & Dowling, 2008). Among the traits of plant growth-promoting endophytes are: ability to fix atmospheric nitrogen, solubilize phosphorus and produce siderophores, certain organic acid and phytohormones like auxins, gibberellins and cytokinins. These plant growth-promoting bacteria (PGPB) also produce certain antioxidants that remove stress induced oxidants and prevent cell damage, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase that minimizes stress-induced ethylene level, thereby, improves root and shoot elongation during stress (Jha, Gupta, Jha, & Mehrotra, 2013).
Furthermore, these bacteria may possess carbonic anhydrase (CA) that have ability to capture the atmospheric carbon dioxide. CA is the primary enzyme for carbon dioxide concentrating mechanism, involved in photosynthesis where it facilitates the reversible conversion of carbon dioxide (CO 2 ) to bicarbonate (HCO 3 − ) at carboxylation site in chloroplast (Lazova, Naidenova, & Velinova, 2004). It regulates the diffusion of CO 2 for phosphoenol pyruvate carboxylase (PEPC) in C4 plants and ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) in C3 plants (Tiwari, Kumar, Singh, & Ansari, 2005). An uncatalyzed interconversion between CO 2 and HCO 3 is 10 4 times slower in comparison to CO 2 flux in photosynthesis (Badger & Price, 1994). Moreover, CA activity is required for CO 2mediated regulation of stomata in guard cells and may provide protection to plant against stress conditions. Any change in plant CA activity directly influences CO 2 assimilation under CO 2 -limiting environment. So, it would be interesting to enhance the photosynthetic CO 2 assimilation in C3 plants by artificial regulation of CA expression (Sun, Wu, Sun, Wu, & Wen, 2014). Wheat is classified as C3 plant and its growth is badly affected due to drought stress. Moreover, competition of CO 2 and O 2 at the site of Rubisco causes loss of fixed carbon. Drought-tolerant endophytic bacteria containing CA may provide promising approach for plant protection against drought stress by enhancing photosynthetic rate. Therefore, present study has been conducted to investigate the impact of endophytic bacteria containing CA on growth and physiological attributes of wheat under water-deficit stress.

Plant material and isolation of culturable endophytic bacteria
Healthy and disease-free wheat plants were randomly collected from five different locations at University of Agriculture, Faisalabad. For isolation of endophytic bacteria, each plant was separated into different parts i.e. root, shoot and leaves and washed thoroughly with tap water to remove adhering dust particles. Plant samples were surface sterilized by dipping in 70% ethanol for 30 s, 3% sodium hypochlorite (NaClO) for 3 min and washing with sterilized distilled water. After sterilization, samples were crushed and serially diluted in 0.85% sodium chloride (NaCl) solution. One hundred microliters from appropriate dilutions were plated in triplicate on two different media, viz Tryptic Soy Agar (TSA) and Luria Bertani (LB). Plates were placed in an incubator at 28ºC for 3-7 days to recover bacterial endophytes. Colonies that appeared morphological different were selected, purified and preserved in glycerol stock at −80ºC for long-term use. Sterility check was also performed to confirm the absence of any bacteria after sterilization by plating the last washing of plant samples on both media.

Drought tolerance ability
Total 150 isolates were tested for drought tolerance by using different levels of PEG-6000 in LB media. Isolates were grown in 100 mL conical flasks containing 50 mL LB media and left for 3 days in shaking incubator at 28ºC and 100 rpm. Bacterial cells were harvested by centrifugating the culture at 4000 × g for 15 min and uniform cell density (10 7 -10 8 CFU mL −1 ) was maintained in LB media. Freshly prepared bacterial culture (0.5 mL) was inoculated into test tubes containing 7 mL LB media with different osmotic potentials and left for 3 days at shaking incubator. Osmotic potential of −0.31, −0.61, −1.09, −1.91 and −3.20 MPa were developed by adding different level of polyethylene glycol (0, 10, 20, 30 and 40%) in LB broth media measured by Cryoscopic Osmometer (OSMOMAT-030-D, Gonotec, Germany). Osmotic potential was measured before and after autoclaving the LB media. Uninoculated control was also maintained at same osmotic potentials with three repeats. Drought tolerance ability was measured by spectrophotometer at 600 nm after 3 days.

CA activity
Fifty drought-tolerant isolates were further tested for CA activity following the method described by Achal and Pan (2011) and Zhang et al. (2011) with some modifications. Bacterial isolates were grown in LB broth and kept at 28ºC for 3 days. Then, these isolates were inoculated in flask containing 100 mL CAproducing medium [yeast extract, 0.7 g; KNO 3 , 0.3g; (NH 4 ) 2 SO 4 , 0.3 g; MgSO 4, 0.05 g; Na 2 MoO 4 , 0.05 g; Na 2 B 4 O 7 .10H 2 O, 0.1 g; Glycerol, 40 mL; 3.5-dintrosalicyclic acid, 0.02 g dissolved in 1 liter] and incubated at 32ºC in mechanical shaker at 150 rpm for 4 days. Cell cultures of bacterial isolates were centrifuged at 8000 × g for 10 min and cell pallets were suspended in Tris-EDTA (pH 8.0) buffer containing 0.01 mg of RNaseI/mL, and kept at 37ºC for 1 h. These bacterial lysates were centrifuged and supernatant was used as CA enzyme solution. The assay mixture consisted of 0.8 mL tris buffer (pH 7.5), 0.1 mL enzyme and 1 mL 4-Nitrophenyl acetate (p-NPA) dissolved in acetonitrile. The released p-nitrophenol was determined at 400 nm by spectrophotometer using p-nitrophenol as standard. Distilled water was used as a blank. One unit enzyme activity represented the amount of enzyme to produce 1 μmol p-nitrophenol per mL.

Plant growth promotion assay
Ten drought-tolerant isolates with higher CA activity were used for plant growth promotion assay. Seeds of wheat variety (Uqab-2000) were surface sterilized with 70% ethanol for 30 s and 3.5% NaClO for 5 min followed by four washings with sterile distilled water. Sterilized seeds were imbibed for overnight in sterilized distilled water to germinate. Four pre-germinated surface-sterilized seeds were dipped in inoculum for 10 min and placed in sterile growth pouch containing half-strength Hoagland solution. Inoculum was prepared in LB media and sterilized broth was used for control. The experiment was conducted with three replicates. Water-deficit stress was induced by dissolving various amounts of PEG-6000 into half strength Hoagland solution to develop −0.04, −1.09 and −1.23 MPa. After 5 days of germination, plants were irrigated with PEG-containing Hoagland solutions for inducing water-deficit stress. The growth chamber was maintained at 25 ± 1 ºC and light and dark period was adjusted at 10 and 14 h, respectively. Light intensity of 275 µmol m −2 s −1 was maintained. Plants were harvested after 21 days and data regarding maximum root length, shoot length, root dry biomass, and shoot dry biomass were collected. Data regarding shoot and maximum root length were measured with the help of meter rod (Khan, Ahmad, Singh, & Nazar, 2006). Dry biomass was determined by drying the sample at 72ºC for 72h.

Physiological parameters
Physiological parameters such as CO 2 assimilation rate (A), transpiration rate (E), stomatal conductance (g s ) and substomatal CO 2 concentration (C i ) were measured 20 days after sowing on fully expanded leaves of wheat seedling using CIRAS 3(PP system, Amesbury, MA, USA) between 10:00 a.m and 2:00 p.m. Intact leaves from each treatment were selected for photosynthetic measurements. The gas-exchange analysis was performed at 25 ± 1ºC temperature and 50 ± 3% relative humidity. The reference level of CO 2 was 380 µmol mol −1 and light intensity (photosynthetic active radiation) was 250 µmol m −2 s −1 . Chlorophyll content was measured using chlorophyll meter (SPAD-502, Minolta, Japan).

Relative water content (RWC) and electrolyte leakage (EL)
For relative water content, fresh leaf samples were weighed and placed in distilled water for 24 h in refrigerator at 4ºC. Then, turgid weight was measured. Dry weight of leaf samples was also noted after placing them in an oven for 24 h. The leaf relative water content was determined following equation as described by Teulat, Zoumarou-Wallis, Rotter, Bahri, and This (2003).
%RWC ¼ ðFresh weight À Dry weightÞ ðFully turgid weight À Dry weightÞ Â100 Leaf disc of plants from each treatment were transferred into test tubes containing 5 mL deionized water to determine electrolyte leakage. Test tubes were placed in shaking incubator (25 ºC) for 4 h at 150 rpm and electrical conductivity (EC) was recorded with EC meter (Jenway Conductivity Meter Model 4070). Then, same tubes were autoclaved at 121ºC for 20 min and EC was determined. Electrolyte leakage was determined with following equation.

CA activity in plant
The CA activity was determined following the method described by Dwivedi and Randhawa (1974). Leaf samples were cut, dipped in cystein hydrochloride solution and incubated for 20 min at 4ºC. The leaf samples were blotted and transferred to test tube containing 0.2 M phosphate buffer (pH = 6.8) followed by 0.2 M alkaline bicarbonate (NaHCO 3 ) and 0.002% bromothymol indicator. These test tubes were incubated at 4ºC for 20 min and titrated against HCl using methyl red as indicator. Results are expressed as mol (CO 2 ) kg −1 Fresh Mass (F.M.) s −1 .

Phenotypic and biochemical characterization of endophytic bacteria
Selected bacterial isolates were streaked on LB agar plates and placed overnight at 28ºC. After 24 h, color and shape of colony was observed. Catalase activity was determined by spreading a loopful culture on glass slide. After spreading, a drop of 35% H 2 O 2 was placed by following the Macfaddin (1980) method. Production of bubbles was considered as positive for catalase. Oxidase activity was performed by rubbing a bacterial culture on filter paper containing Kovacs reagent. Change in color from blue to purple was considered positive for oxidase (Steel, 1961).

Growth promoting traits of selected endophytic bacteria
Indole-3-acetic acid production was observed with and without L-tryptophan using Salkowski reagent and measured at 535 nm by spectrophotometer and indole acetic acid production was calculated by standard curve (Sarwar, Arshad, Martens, & Frankenberger, 1992). Ability of bacterial strains to solubilize phosphate was determined by spotting 10 µL of overnight culture of each strain on NBRI-PBP media (Mehta & Nautiyal, 2001). For hydrocyanide production (HCN), isolates were grown in King B medium supplemented with 4.4 gL −1 glycine (Lorck, 1948). Hydrocyanic acid was observed with change in color of filter paper previously dipped in sodium carbonate solution prepared in picric acid from yellow to brown. Ability of bacterial isolates to produce chitinase was observed in LB media supplemented with chitin (Chernin et al., 1998). Loopful amount of inocula was placed on four different places in Petri plates and put in incubator at 28 ± 1ºC. After 3 days of incubation, halo zone was observed around the colonies, where presence of halos was indicator for chitinase activity. Similarly, loopful of bacterial culture was placed at four places on Petri plates containing RCV-glucose media (Ashraf, Berge, & Mahmood, 2004). Plates were examined for mucoid growth after 96 h of incubation and colonies showing mucoid growth were considered positive for exopolysaccharides (EPS) production.

Identification of efficient isolates
Efficient endophytic bacterial isolates (WR2, WS11 and WL19) on the basis plant growth stimulation were selected for identification. These isolates were identified by 16S rRNA gene sequencing. Sequencing process of bacterial isolates was performed by Macrogen Inc. (Korea). The partial sequences of nucleotide were analyzed using Basic Local Alignment Search Tool (BLAST) program on National Center for Biotechnology Information site and identified on the basis of closest homology.

Statistical analysis
Data were analyzed according to completely randomized design (Steel, Torrie, & Dicky, 1997) whereas difference among the means was analyzed by Tukey test using Statistic 8.1 software.

Endophytic bacterial isolates and their drought tolerance ability
A total of 150 endophytic bacterial isolates from different tissues of wheat plants were isolated using LB and TSA media. After isolation, these isolates were cultured in LB medium at different osmotic potentials to assess their survival ability. Out of 150 isolates, only 50 were able to grow at osmotic potentials ranging from −0.31 to −3.20 MPa. Among the 50 isolates, 10 isolates WR2, WS7, WS11, WS22, WS23, WL9, WL13, WL16, WL19 and WL20 which possessed higher CA activity are shown in Table 1 for their growth and survival ability. Out of 10 isolates, isolate WR2, WS11, WL13 and WL19 showed higher drought tolerance ability at any PEG concentrations compared to other isolates. However, their optical density decreased with increasing the PEG-induced osmotic stress.

CA activity
Fifty bacterial isolates showing tolerance to drought were further screened for CA activity (Supplemental data). Among the 50 bacterial isolates, 10 isolates which showed higher CA activity of are given in Figure 1. Maximum CA activity was observed by bacterial endophytes WL19, WS11 and WS23 and followed by WR2 and WL9, respectively. Results also revealed that isolates (WR2, WS11 and WL19) having drought tolerance ability showed better CA activity.

Effect of endophytic bacteria on plant growth
Ten drought-tolerant CA-containing endophytic bacterial isolates were tested for improving growth and physiology of wheat under water-deficit stress.
Results showed that water deficit caused significant reduction in maximum root length but inoculation with drought-tolerant CA-containing endophytic bacteria enhanced maximum root length compared to uninoculated control. All the isolates showed significant increase in maximum root length under non-stressed as well as stressed conditions (Table 2). Isolates WR2, WS11 and WL19 performed significantly better compared to others and uninoculated control under non-stressed (−0.04 MPa) as well as stressed conditions (−1.09MPa). However, isolate WL19 showed significant increase in maximum root length compared to other isolates under severe water-deficit conditions (−1.23 MPa). Highest increase up to 65.0% in maximum root length was recorded by the inoculation of isolate WL19 under PEG-induced water-deficit stress (−1.23 MPa) compared to respective control. Inoculation with endophytic bacterial isolates significantly improved the   shoot length under non-stressed as well as stressed conditions (

Effect of bacterial endophytes on physiological attributes
Chlorophyll content, CA activity, net photosynthetic assimilation, stomatal conductance and transpiration rate was also determined in wheat plant. Water-deficit stress significantly decreased the leaf chlorophyll content but inoculation with bacterial isolate WL19 showed 39.1 and 48.5% increase at −0.04 and −1.09 MPa, respectively (Table 3). However, under severe waterdeficit conditions (−1.23 MPa), bacterial isolate WL9 showed 57.2% increase in chlorophyll compared to control plants followed by WL19 and WS11, respectively. CA activity was also improved in bacterial inoculated plants under non-stressed as well as stressed conditions. Isolates WR2, WS11 and WL19 significantly enhanced CA activity compared to uninoculated control under non-stressed (−0.04 MPa) as well as stressed conditions (−1.09, −1.23 MPa) (Table 3). Inoculation with isolate Table 3. Effect of drought-tolerant CA-containing endophytic bacterial isolates on root dry weight, shoot dry weight, chlorophyll content and carbonic anhydrase activity of wheat under non-stressed and PEG-induced water-deficit conditions. WR2, WS11 and WL19 showed significant increase in CA activity up to 59.5, 64.6 and 68.2%, respectively, compared to uninoculated control under water-deficit conditions (−1.23 MPa). Water-deficit stress also decreased the net photosynthetic assimilation, stomatal conductance and transpiration efficiency but inoculation with bacterial endophytes improved these physiological parameters under mild as well as severe water-deficit conditions (−1.09, −1.23 MPa). Isolate WR2, WL19, WS11 and WS23 showed significant increase in photosynthetic rate compared to other isolates and uninoculated control (  (Table 4).
However, at PEG-induced water-deficit stress (−1.09MPa) isolate WR2 showed significant increase up to 44.4% compared to others. Isolate WL19 improved stomatal conductance by 60.3% under PEG-induced water-deficit conditions of −1.23 MPa (Table 4). Substomatal CO 2 concentration was significantly decreased by the inoculation of bacterial isolate WR2, WS11 and WL19 under non-stressed and stressed conditions. Isolate WL19 showed 47.2% decrease in substomatal CO 2 concentration under water-deficit conditions (−1.23MPa) compared to respective control (Table 4). Results also showed the significant positive correlation between bacterial CA and photosynthesis under non-stressed as well as stressed conditions (Figure 2).

Relative water content, electrolyte leakage
Water-deficit conditions significantly decreased the RWC and increased the electrolyte leakage in noninoculated plants (  in wheat plant (Table 5). However, inoculation with endophytic bacterial isolates caused significant reduction in electrolyte leakage under PEG-mediated waterdeficit conditions (−1.09, −1.23MPa). Maximum electrolyte leakage was observed in control while minimum was observed in inoculated plants in severe water-deficit stress (−1.23 MPa). Isolate WL19 showed 37.3% inhibition in electrolyte leakage followed by WS11, WL13 and WR2 under severe water-deficit stress (−1.23MPa) compared to respective control plants.

Characterization and identification of efficient isolates
Plant growth-promoting characteristics of efficient endophytic bacterial isolates (WR2, WS11, WL19) are shown in Table 6. These isolates were positive for catalase activity. Isolate WL19 was positive while isolates WR2 and WS11 were negative for oxidase test. Isolates WR2, WS11 and WL19 were able to solubilize the tricalcium phosphate. These isolates produced IAA from 10.94 to 73.7 μg mL −1 in the presence as well as absence of L-tryptophan and maximum IAA production was observed by bacterial isolate WL19. These isolates were also found positive for HCN production and chitinase activity. Moreover, EPS production was observed in all isolates. These isolates were identified by 16S rRNA as Bacillus marisflavi (WR2) Bacillus thuringiensis (WS11) and Bacillus subtilis (WL19).

Discussion
Plants under natural conditions are exposed to several biotic and abiotic stresses where water deficit is a major   constraint to plant growth and crop productivity affecting agricultural production. Endophytic bacteria have recently been reported for their potent role in improving the plant growth under stressed environment. However, potential of drought-tolerant endophytic bacteria to alleviate drought stress in plant is least explored. In the present study, potential of bacterial endophytes to adapt to drought stress were assessed by growing them at −0.31, −0.61, −1.09, −1.91 and −3.20 MPa osmotic potential with PEG-6000 induced drought stress. Among the 150 isolates, 50 isolates showed highest growth at different PEG concentrations. However, their growth decreased with increasing PEG concentration. These drought-tolerant endophytic bacteria also contained high CA activity. Out of 50, ten isolates which showed highest CA activities were selected (Figure 1). These isolates also had ability to tolerate highest level of drought stress (Table 1). These finding are in accordance to the observation of previous reports where PGPB showed growth at −0.73 MPa (Vardharajula et al., 2011) and Shinorhizobium sp. SK27 and SK36 at −3.5 MPa (induced with PEG) (Abolhasani, Lakzian, Tajabadipour, & Haghnia, 2010). Survival ability of bacteria under stress condition might be due to exopolysaccharides production that protects the microbes from water scarcity by improving water retention and modulating the diffusion of organic carbon (Chenu & Roberson, 1996). Results of present study showed that efficient isolates were found positive for EPS production (Table 6). Moreover, ability to produce oxidase, catalase and exopolysaccharide by bacteria has also been attributed to their survival under severe water-deficit condition (Goyal, Chetal, & Nainawatee, 1986). The results of current study also showed that isolates WR2, WS11 and WL19 were positive for catalase production while isolate WL19 was positive for oxidase test (Table 6). Plant growth and development is reduced under drought condition due to disruption in biochemical and physiological processes. Microbial inoculants can help plants to sustain their growth under stress environment. In the present study, inoculation with endophytic bacteria improved plant growth by increasing maximum root and shoot length (Table 2), root and shoot dry weight (Table 3) under non-stressed as well as stressed conditions. Increase in root growth has been reported by inoculation of PGPB in many plants (Dawwam, Elbeltagy, Emara, Abbas, & Hassan, 2013;Delshadi, Ebrahimi, & Shirmohammadi, 2017). Synthesis of auxins by bacteria affects the root system through increasing the size and number of the adventitious roots (Gutierrez et al., 2012) resulting in improved water and nutrient uptake that may have positive influences on plant growth. In this study, endophytic bacterial isolate WL19 produced IAA of 73.7 μg mL −1 (Table 6) and showed maximum root growth compared to other isolates. Ability of bacterial isolates to produce IAA reveals their potential for use as growth promoter (Majeed, Abbasi, Hameed, Imran, & Rahim, 2015). Improved phosphorus nutrition has been proposed to influence root development and plant growth (Jones & Darrah, 1994). Our investigation showed isolate WR2, WS11 and WL19 have ability to solubilize tricalcium phosphate Ca 3 (PO 4 ) 2 (Table 6). Of the 10 isolates evaluated in wheat, isolates WR2, WS11 and WL19 significantly enhanced shoot biomass compared to control plants. Increase in shoot weight might be due to CA activity of endophytic bacteria that supplied higher photoassimilate to plant by increasing the photosynthetic rate. There could be several possible reasons of increasing growth as has been found in other bacterial endophytes such as hormone production, enhanced phosphorus utilization, increased nitrogen fixation and iron acquisition (Hardoim, Overbeek, & Elsas, 2008). Researchers have also reported that plant biomass increased up to 78% in bacterially primed plant under drought stress (Timmusk et al., 2014).
In the present study, inoculation with endophytic bacterial isolates improved chlorophyll content, CA activity (Table 3), photosynthesis, stomatal conductance, substomatal CO 2 concentration and transpiration rate (Table 4) compared to un-inoculated control plants. These results are in line with Shi, Lou, and Li (2010) where endophyte-infected sugar beet showed significant increase in carbon assimilation, chlorophyll content, stomatal conductance and transpiration rate. Increase in photosynthetic activity was due to increase in cholorophyll content that consequently enhanced the carbohydrate synthesis, might be due to enhanced metabolism of chloroplast by endophytic infection. Another possible explanation can be bacterially produced CA. However, role of bacterial produced CA in plant photosynthesis is not understood. This enzyme is involved in several physiological processes such as photosynthesis and CO 2 transport (Lazova et al., 2004) and regulates the stomatal conductance by maintaining equilibrium between CO 2 and HCO 3 (Tiwari et al., 2005). Results also showed that photosynthetic rate was significantly correlated with CA activity in bacteria under non-stressed as well as stressed conditions. However, correlation was more positive under stressed conditions (Figure 2). It has been reported that CA activity was inhibited in ethoxyzolamide-treated C3 plant and caused 80-90% reduction in photosynthesis at low CO 2 concentration showing the key role of CA (Badger & Pfanz, 1995). In addition, Perez-Martin et al. (2014) studied the role of CA enzyme in the stomatal and mesophyll conductance and found that CA enzyme had small but important role in stomatal conductance in 5-year-old olive plant under water stress. Furthermore, CA activity is required for CO 2 regulated stomatal opening and closing and can be alternative approach to provide protection against unfavorable conditions (Sun et al., 2014).
Relative water content decreased under stressed and non-stressed conditions but inoculation with bacterial endophytes improved the relative water content ( Table 5). The relative water content is considered an indicator of plant water-deficit stress tolerance. Decrease in RWC has been observed in many plants under drought stress. Inoculated plants minimize the inhibitory effect of water stress on plant root and develop more effective root system (Dodd et al., 2010). In contrast to RWC, water-deficit stress also stimulated the electrolyte leakage in inoculated and uninoculated plants but inoculation with endophytic bacterial isolates helped to reduce the damage under water-deficit stress compared to uninoculated seedlings. Vardharajula et al. (2011) found that a direct correlation exists between membrane damage and drought sensitivity; bacterial inoculation decreased the membrane damage caused by water-deficit stress.
Moreover, increase in growth and physiological parameters by the application of drought-tolerant CA-containing endophytic bacteria was more with bacterial inoculation compared to uninoculated control under non-stressed and stressed conditions (Table 7).

Conclusion
This study provides an evidence that drought-tolerant endophytic bacteria having higher CA activity enhanced plant growth under water-deficit stress. Based on our results, we conclude that endophytic bacterial isolates WR2, WS11 and WL19 improved seedling growth, physiology and relative water content more efficiently compared to uninoculated control under non-stressed as well as stressed conditions. We have also demonstrated that these bacteria possess characteristics i.e. IAA production and phosphate solubilization which are considered important plant growthpromoting traits. Multi-site experiments are needed to further evaluate potential of CA-containing endophytic bacteria under natural/field conditions.