research paper
research paper
Plant Signaling & Behavior 8:10, e26891; October; © 2013 Landes Bioscience
Piriformospora indica rescues growth diminution of rice seedlings during high salt stress Abhimanyu Jogawat1, Shreya Saha1, Madhunita Bakshi3, Vikram Dayaman1, Manoj Kumar1, Meenakshi Dua2, Ajit Varma3, Ralf Oelmüller4, Narendra Tuteja5, and Atul Kumar Johri1,* 1 School of Life Sciences; Jawaharlal Nehru University; New Delhi, India; 2School of Environmental Sciences; Jawaharlal Nehru University; New Delhi, India; 3Amity Institute of Microbial Technology; Amity University; Noida, UP, India; 4Institute of Plant Physiology; Friedrich-Schiller-University; Jena; Jena, Germany; 5Plant Molecular Biology Group; International Center for Biotechnology and Genetic Engineering; Aruna Asaf Ali Marg, New Delhi, India
Keywords: Arbuscular mycorrhizal fungi, Piriformospora indica, root endophytic fungus, salinity stress tolerance Abbreviations: Chlorophyll (chl), Piriformospora indica (P. indica)
Piriformospora indica association has been reported to increase biotic as well as abiotic stress tolerance of its host plants. We analyzed the beneficial effect of P. indica association on rice seedlings during high salt stress conditions (200 and 300 mM NaCl). The growth parameters of rice seedlings such as root and shoot lengths or fresh and dry weights were found to be enhanced in P. indica-inoculated rice seedlings as compared with non-inoculated control seedlings, irrespective of whether they are exposed to salt stress or not. However, salt-stressed seedlings performed much better in the presence of the fungus compared with non-inoculated control seedlings. The photosynthetic pigment content [chlorophyll (Chl) a, Chl b, and carotenoids] was significantly higher in P. indica-inoculated rice seedlings under high salt stress conditions as compared with salt-treated non-inoculated rice seedlings, in which these pigments were found to be decreased. Proline accumulation was also observed during P. indica colonization, which may help the inoculated plants to become salt tolerant. Taken together, P. indica rescues growth diminution of rice seedlings under salt stress.
Introduction
Results
P. indica, a root endophytic filamentous fungus, resides in plant root cortical cells.1 P. indica has been classified in the Sebacinales order. The most intriguing character of this beneficial fungus is that it has a broad host spectrum range that includes monocots, eudicots, and dicots.1-10 P. indica has been shown to improve the plant growth, nutrient uptake, and helped plants during abiotic and biotic stress tolerance. Because of these virtues, P. indica has proven to be a plant biofertilizer, probiotic, and biohardening tool.11-13 P. indica-mediated salt tolerance mechanism was found to be linked strongly with increase in antioxidants under salt stress in barley which attenuates the NaCl-induced lipid peroxidation, metabolic heat efflux, and fatty acid desaturation in barley leaves.10 It has been reported that P. indica significantly elevated the amount of ascorbic acid and increased the activities of antioxidant enzymes in barley roots during salt stress conditions.14 In response to high salt stress, plants have developed a number of mechanisms to counteract high salt stress, such as mineral ion homeostasis and accumulation of solutes such as proline.15 In the present study we analyzed the growth parameters, photosynthetic pigment, and proline contents of P. indica-inoculated and noninoculated rice seedlings under high salt stress.
P. indica colonization P. indica colonization of rice roots was checked after 15 and 20 d of inoculation (dpi). We have observed colonization at 15 and 20 dpi; however, no colonization was observed in plants that were inoculated using autoclaved P. indica. In plants that were inoculated with the living fungal chlamydospores, we have observed 60 and 70% colonization of the entire root at 15 and 20 dpi, respectively (Fig. 1A-C). P. indica association improves growth of rice plants We have found that roots of P. indica-inoculated rice plants were hard, thick, brownish, and higher in number as compared with the non-inoculated plants (Fig. 2A and B). In general, we have observed that P. indica colonization enhanced root and shoot lengths as well as the fresh and dry weights of the host plants (Table 1). While the difference between the shoot lengths for non-inoculated and inoculated plants was found to be non significantly different, when the plants were treated with 200 mM NaCl (at day 5 and 15) or 300 mM NaCl (at day 15), the difference in the root lengths was significantly different (P < 0.05) at all times points, except at day 15 when 300 mM NaCl treatment was given. At both 200 and 300 mM NaCl treatment, we have observed that P. indica significantly
*Correspondence to: Atul Kumar Johri; Email:
[email protected] Submitted: 09/07/2013; Revised: 10/18/2013; Accepted: 10/21/2013 Citation: Jogawat A, Saha S, Bakshi M, Dayaman V, Kumar M, Dua M, Varma A, Oelmüller R, Tuteja N, Johri AK. Piriformospora indica rescues growth diminution of rice seedlings during high salt stress. Plant Signaling & Behavior 2013; 8:e26891; PMID: 24169531; http://dx.doi.org/10.4161/psb.26891 www.landesbioscience.com Plant Signaling & Behavior e26891-1
brownish, have stunted growth and reduced weight. Chl a, Chl b, and carotenoid content was measured in inoculated and non-inoculated plants at high salinity stress. A strong effect of the high salt stress was seen at 5 dpi. The photosynthetic pigments decreased much stronger in non-inoculated plants as compared with the P. indica-inoculated plants. P. indica colonization results in higher Chl a levels compared with the non-inoculated controls at all time points (Table 2). We have found that Figure 1. P. indica colonization of rice roots. (A) Negative control inoculated with autoclaved P. inoculated plants have significantly (P < indica shows no chlamydspores in the root cortex region. (B) Rice root segment showing coloniza0.05) higher Chl b at 10 dpi as compared tion at 15 dpi. (C) Root colonization at 20 dpi. Fifteen days after P. indica inoculation, 60% colonizawith the non-inoculated plants when tion was detected. Control rice plants were mock treated with autoclaved P. indica and contained 200 mM NaCl treatment was given. no spores. Arrow indicates a single chlamydospore of P. indica. Furthermore, P. indica colonization significantly (P < 0.05) resulted in the higher carotenoid content. A 3.15-fold difference was found in the carotenoid content in plants exposed to 300 mM NaCl at 15 dpi, and this difference was found to be significant (P < 0.05) (Table 2). Progression of P. indica colonization affects proline content of rice plants Proline accumulation is an immediate response of plants to combat any type of stress. We have observed that the proline content increased significantly (1.5-fold, P < 0.05) in P. indica-inoculated rice seedlings as compared with the non-inoculated Figure 2. P. indica colonization. (A) Non-inoculated rice roots were thinner when compared with P. plants when 200 mM NaCl treatment indica-inoculated rice roots. Strong, hard, and brownish roots were observed in P. indica-inoculated was given (Table 3). Interestingly, we rice plants. (B) Root number in 25-d-old control plants and those inoculated by P. indica (15 dpi). also observed an enhanced proline Root numbers are higher in case of P. indica-inoculated rice. Each column represents the means of 3 measurements ± Standard Error. content in P. indica-inoculated rice plants that are not exposed to salt stress (P < 0.05) increased the dry weight of the plants at all time points as compared with the non-inoculated as compared with the non-inoculated plants (Table 1). plants. Without salt stress, an approximately 6-fold difference in P. indica-inoculated plants combat with high salt stress in proline content was detected between P. indica-inoculated and better way control plants. This suggests that P. indica-inoculated seedlings Under high salt conditions P. indica-inoculated rice grows become more salt tolerant because of higher proline accumulation better and remains healthier as compared with the non-inoculated (Table 3). control (Fig. 3A and B). The growth parameters of inoculated rice Discussion plants were significantly improved under high salt stress compared with high salt stressed non-inoculated plants. Growth of nonIn the present study, we used rice plants to show higher inoculated plants was ceased after a certain period of time and the resistibility against salinity stress upon colonization with P. seedlings were found to become brownish in color. Progression of P. indica colonization affects photosynthetic indica. Salinity is one of the major abiotic stress that affects plants growth, development, and productivity. Here, we demonstrate pigment content of rice plant A major response of salinity stress in plants is the degradation that important physiological parameters that are related to growth, of photosynthetic pigments, which is caused by chlorosis, reduced photosynthesis, and stress-induced metabolite production are photosynthesis, and oxidative damage. As a result, plants become impaired under salt stress in rice plants.
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Table 1. Growth parameters of non-inoculated (NI) and P. indica-inoculated (PI) rice
Growth analysis is a fundamental characteristic to seedlings study plant’s response to an environmental stress. Salt No salt stress 200 mM salt stress 300mM salt stress stress induces stomatal closure, declines photosynthesis, induces photoinhibition, and disrupts nutrient balance NI PI NI PI NI PI by affecting the availability, transport, and partitioning of nutrients.16 Thus, an immediate effect of salt Shoot length stress causes lower body mass, stunted growth, and Day 0 10.4 ± 0.1 10.4 ± 0.1 10.4 ± 0.1 10.4 ± 0.1 10.4 ± 0.1 10.4 ± 0.1 reduced chlorophyll concentration. To investigate the Day 5 10.8 ± 0.4 14.5 ± 0.6 11.1 ± 0.2 12.4 ± 0.5* 12.2 ± 0.7 13.7 ± 0.8 contribution of P. indica in protecting the plant against salinity stress, we inoculated rice plants with the Day 10 13.0 ± 0.4 15.2 ± 0.4* 10.9 ± 1.3 13.2 ± 0.6 11.3 ± 0.2 14.1 ± 0.2 fungus. We measured dry and fresh weights, root, and Day 15 13.1 ± 0.5 15.3 ± 0.3 12.4 ± 0.3 13.2 ± 0.3* 12.0 ± 0.4 13.9 ± 2.0* shoot lengths of inoculated plants grown in normal Root length soil and compare the data with the non-inoculated Day 0 8.3 ± 1.0 8.3 ± 1.0 8.3 ± 1.0 8.3 ± 1.0 8.3 ± 1.0 8.3 ± 1.0 plants grown under the same condition. We found that inoculated plants show significantly higher growth Day 5 9.6 ± 0.5 12.7 ± 0.6 8.1 ± 0.1 12.2 ± 0.4 8.3 ± 0.7 11.1 ± 0.9 rates than non-inoculated plants. In our previous study Day 10 9.6 ± 0.5 13.3 ± 0.7 8.7 ± 0.8 12.2 ± 0.5 8.8 ± 1.0 12.5 ± 0.4 by Kumar et al.,17 we also observed a growth promoting Day 15 11.9 ± 1.5 18.1 ± 1.3 13.4 ± 0.5 15.3 ± 0.7 10.5 ± 0.2 13.8 ± 1.2* activity of P. indica on plants by providing essential Fresh weight mineral nutrients, mainly phosphate, to plants. Here, we report for the first time a significant effect of P. Day 0 34.2 ± 0.2 34.2 ± 0.2 34.2 ± 0.2 34.2 ± 0.2 34.2 ± 0.2 34.2 ± 0.2 indica on growth of a salt sensitive variety of rice under Day 5 49.2 ± 2.3 68.0 ± 2.6 50.8 ± 0.6 66.6 ± 1.4 49.5 ± 1.9 53.5 ± 0.4* high salt stress. We found that upon colonization, Day 10 53.9 ± 0.3 68.1 ± 5.0 53.2 ± 0.1 74.1 ± 1.7 64.3 ± 1.9 84.3 ± 1.8 rice plants can withstand up to 300 mM NaCl and Day 15 81.1 ± 3.1 105.0 ± 1.5 78.1 ± 1.5 95.4 ± 0.8 78.4 ± 1.6 108.9 ± 3.5 show significantly higher growth than non-inoculated plants. Root and shoot lengths are also found to be Dry weight higher as compared with non-inoculated plants. Root Day 0 12.6 ± 0.6 12.6 ± 0.6 12.6 ± 0.6 12.6 ± 0.6 12.6 ± 0.2 12.6 ± 0.6 lengths are important parameters for salt stress because Day 5 16.0 ± 0.2 26.2 ± 1.9 15.3 ± 0.2 19.8 ± 1.0 15.7 ± 0.2 20.1 ± 1.0 the roots are in direct contact with soil and absorb Day 10 19.6 ± 0.8 29.8 ± 0.1 16.9 ± 0.2 21.9 ± 1.2 18.4 ± 0.2 22.9 ± 1.1 water from soil.20 Photosynthesis is important for growth and Day 15 22.0 ± 1.3 31.6 ± 0.7 17.9 ± 0.6 26.3 ± 2.3 14.9 ± 0.2 20.3 ± 1.5 development of green plants and can be severely Note: Root and shoot lengths of non-inoculated and P. indica-inoculated rice seedlings affected by environmental stress. In the present study, under high salt stress: Root and shoot lengths were found high as colonization progressed. we focused on Chl and carotenoid levels, since they Fresh weight and dry weight of rice seedlings grown under high salt stress: Fresh and dry play a vital role in photosynthesis and photoprotection. weights were found to be high in P. indica-inoculated plants. Each data set represents the Chl and carotenoids can be degraded due to high means of 3 independent measurements ± SE *indicates not significant as compared with the control (non-inoculated); all other data are found significant at P < 0.05. sodium ion toxicity during salt stress. Several crops have been reported of having reduced Chl a and Chl b concentrations upon salinity stress, e.g., cabbage (Brassica oleracea during salinity stress causes the disruption of osmotic balance, capitata L.), sunflower (Heliantus annuus L.), wheat (Triticum membrane disorganization, reduced enzyme activities, breakdown aestivum L.), and sugarcane.18-24 The extent of reduction of the of protein, and metabolic toxicity inside the plant cell. Osmolytes pigment content depends on the salt tolerance of the plant species. are considered as the major component to maintain osmotic In case of some plant species (wheat, pea, sunflower, etc.), the balance inside the cell and protect plants from potential toxic Ch content is a potential biochemical indicator of salt tolerance, damage. When exposed to stressful conditions, plants accumulate although this is not true for all species and cultivars.23 In a study an array of metabolites, particularly amino acids. Proline, an with sugarcane, Gomathi and Rakkiyapan24 found that salt stress amino acid, plays a highly beneficial role in plants exposed to at various plant growth stages caused a marked reduction in various stress conditions. Besides acting as an excellent osmolyte, Chl and carotenoid contents, but salt-tolerant varieties exhibited it can also work as a metal chelator, an antioxidative defense higher membrane stability and pigment contents. We measured molecule, and a signaling molecule. Stressful environment results the content of Chl a, Chl b, and carotenoids in 200- and 300- in an overproduction of proline in plants, which in turn imparts mM salt-treated plants with P. indica inoculation. The amount stress tolerance by maintaining cell turgor or osmotic balance, by of these pigments was significantly higher in inoculated plants as stabilizing membranes thereby preventing electrolyte leakage, and compared with the non-inoculated control grown under the same by maintaining the reactive oxygen species level within normal salt conditions. Therefore, P. indica helps rice plants by protecting ranges, thus preventing oxidative burst in plants.25 In this study them against salt stress. we have observed that proline accumulated significantly higher The most detrimental role of salinity stress in plants is high ion in inoculated rice plants as compared with non-inoculated toxicity and osmotic misbalance. High level of ion accumulation plants during the high salt treatment. Plants inoculated by P.
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indica survived better under high salt treatment, which indicates that P. indica is helping the plant in maintaining the osmotic pressure. However, it needs more detailed studies to completely understand the mechanism of how P. indica is helping the plant in maintaining osmotic balance. Most studies concerned with arbuscular mycorrhiza-saline soil interactions do not simulate field conditions.26 Our study supports the idea of using and testing P. indica in field conditions.
Materials and Methods Plant and fungal culture and growth conditions Seeds of rice (Pusa basmati-1, IARI) were surface-sterilized for 15 min in ethanol followed by dipping for 20 min in a 4% NaClO solution, and finally washed 6 times with sterile water. Seeds were cold treated Figure 3. Rice plants after 10 d salt stress. For representation purposes, one pot per treatment is shown. and germinated on water-agar plates (A) For 200 mM and (B) for 300 mM salt stress. (a) Non-inoculated rice without salt treatment. (b) P. indica(0.8% Bacto Agar, Difco) at 25°C inoculated rice without salt treatment. (c) Non-inoculated rice treated with 200 (300) mM salt. (d) P. indicain the dark for 3 d. Seedlings were inoculated rice treated with 200 (300) mM salt. placed in pots (9 cm height by 10 cm diameter) containing sand (2–4 mm Table 2. Photosynthesis pigments of non-inoculated (NI) and P. indica-inoculated (PI) rice seedlings diameter). Plants were weekly supplied with No salt stress 200 mM salt stress 300mM salt stress half-strength modified Hoagland solution NI PI NI PI NI PI containing the following composition: 5 mM Chl a content KNO3, 5 mM Ca(NO3)2, 2 mM MgSO4, 10 µM KH2PO4, 10 μM MgCl2, 4 μM ZnSO4, 1 Day 0 0.35 ± 0.01 0.35 ± 0.01 0.35 ± 0.01 0.35 ± 0.01 0.35 ± 0.01 0.35 ± 0.01 μM CaSO4, 1 μM NaMoO4, 50 μM H3BO3. Day 5 0.35 ± 0.01 0.76 ± 0.05 0.55 ± 0.01 0.85 ± 0.05 0.31 ± 0.02 0.57 ± 0.01 P. indica was cultured on Aspergillus minimal Day 10 0.44 ± 0.02 0.86 ± 0.04 0.34 ± 0.02 0.49 ± 0.03 0.26 ± 0.02 0.50 ± 0.01 media for 7 d as described previously.27 Day 15 0.46 ± 0.02 0.90 ± 0.00 0.07 ± 0.01 0.16 ± 0.03 0.04 ± 0.02 0.22 ± 0.03 P. indica colonization Three-day-old germinated seedlings were Chl b content planted in pots without P. indica and allowed to Day 0 0.14 ± 0.02 0.14 ± 0.02 0.14 ± 0.02 0.14 ± 0.02 0.14 ± 0.02 0.14 ± 0.02 grow for 7 d. Rice seedlings were taken out, and Day 5 0.14 ± 0.01 0.22 ± 0.02* 0.20 ± 0.02 0.26 ± 0.02* 0.19 ± 0.01 0.23 ± 0.01* the roots were washed and were inoculated with Day 10 0.17 ± 0.01 0.25 ± 0.02 0.14 ± 0.03 0.20 ± 0.02 0.14 ± 0.01 0.24 ± 0.02* P. indica with sterile sand mixed with the fungal culture (1% fungus in sand by w/w). Control Day 15 0.19 ± 0.01 0.33 ± 0.02* 0.06 ± 0.02 0.08 ± 0.03* 0.03 ± 0.01 0.08 ± 0.02* plants were mock inoculated with autoclaved Carotenoid content (dead) P. indica-mixed sand. Plants were given Day 0 31.6 ± 0.16 31.6 ± 0.16 31.7 ± 0.16 31.6 ± 0.16 31.6 ± 0.16 31.6 ± 0.2 half strength Hoagland’s nutrient solution Day 5 35.5 ± 0.59 52.09 ± 1.12 41.4 ± 0.07 47.3 ± 0.69 37.7 ± 1.8 41.9 ± 0.6 weekly. P. indica colonization was checked 15 and 20 dpi under the light microscope (Leica Day 10 36.0 ± 0.12 52.9 ± 1.46 24.3 ± 2.22 39.4 ± 0.01 21.1 ± 1.3 36.15 ± 1.3 type 020–518.500) as described previously.28 In Day 15 36.2 ± 0.40 58.7 ± 0.23 7.7 ± 0.83 14.8 ± 2.70 5.4 ± 1.0 17.0 ± 1.9 brief, colonization was checked by taking 10 root Note: Photosynthetic pigment content of non-inoculated and P. indica-inoculated rice plants: samples randomly from 3 different inoculated The values obtained by 3 independent samples were divided by leaf FW, and the photosynthetic rice plants 15 dpi and 20 dpi, i.e., when the rice pigment content was calculated as nmol/ml/mg of leaf FW. Each data set represents the means seedlings were 25 and 30 d old, respectively. of 3 independent experiments ± SE. The pigment concentrations were more decreased in nonSamples were softened in 10% KOH solution inoculated plants. *indicates not significant as compared with the respective control; all other data are found significant at P < 0.05. e26891-4 Plant Signaling & Behavior
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for 15 min, acidified with 1 M HCl for 10 min, and finally stained with 0.02% Trypan blue overnight.28-30 Samples were distained with 50% lacto-phenol for 1–2 h prior to observation under the light microscope. The distribution of intracellular chlamydospores within the cortex region of root was taken as an indication of colonization.8 The percentage colonization of the full length root was calculated for the inoculated plants according to a published method.31 Salt treatment and growth analysis of rice Salt treatment was given when rice seedlings were 10 d old. In case of P. indica-inoculated plants, salt treatment was given in parallel. Four sets of plants were analyzed in this study to check the role of P. indica during salt treatment: 1) Non-inculated plants without salt treatment, 2) plants inoculated with P. indica and salt treatment, 3) plants inoculated with P. indica without salt treatment, and 4) non-inoculated plants with salt treatment. In case of P. indica-inoculated plants, salt stress was given at day 10, and this day was taken as day 0 of salt stress. Two-hundred – and 300-mM NaCl solutions were prepared in 1 × half strength Hoagland solution. Pots having 6–10 plants were placed in trays with different salt solutions.32 Rice root numbers and length, shoot length, fresh and dry weight of P. indica-inoculated and noninoculated plants were measured after 5, 10, and 15 d of parallel salt treatments. Determination of chlorophyll a, b and carotenoids contents Leaves were harvested, weighed, and ground in 90% ammonical acetone (acetone: water: 0.1 N ammonia, ratio of 90: 9: 1) at 4°C. Pigments contents were measured at 663, 645, and 470 nm for Chl a, Chl b, and carotenoids, respectively, using the supernatant. Total Chl content was measured by spectrophotometer and calculated as nmol/ml.33 Chl a = (14.21 × OD663 – 3.01 × OD645), Chl b = (25.23 × OD645 – 5.16 × OD663), Chl (a+ b) = (9.05 × OD663 + 22.2 × OD645), and carotenoids = {1000 × OD470 – (3.27 × Chl a – 1.04 × Chl b)/5}. The obtained values were divided by leaf fresh weight to obtain values in nmol/ml/mg of leaf fresh weight. Proline content measurement Proline accumulation is one of the immediate plant responses to stress, therefore the proline content was measured at day 0, 1, 2, 4, 5, 10, and 15. Plants were 10, 11, 12, 14, 15, 20, and 25 d old at these measuring points when P. indica inoculation or salt treatment or both treatments or none of them were applied. Proline content was determined according to method described previously.34 In brief, 0.5 g of plant material was homogenized in 10 ml of 3% aqueous sulfosalicylic acid and the homogenate was centrifuged References 1.
2.
Verma S, Varma A, Rexer K, Hassel A, Kost G, Sarbhoy A, Bisen P, Bütehorn B, Franken P. Piriformospora indica, gen. et sp. nov, a new root-colonizing fungus. Mycologia 1998; 90:896-903; http://dx.doi. org/10.2307/3761331 Peskan-Berghöfer T, Shahollari B, Giong PH, Hehl S, Markert C, Blanke V, Kost G, Varma A, Oelmüller R. Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant-microbe interactions and involves early plant protein modifications in the endoplasmic reticulum and at the plasma membrane. Physiol Plant 2004; 122:465-77; http://dx.doi. org/10.1111/j.1399-3054.2004.00424.x
3.
4.
Table 3. Total proline content of non-inoculated (NI) and P. indicainoculated (PI) rice seedlings under 200 mM salt stress (SS) NI
PI
NI+SS
PI+SS
Day 0
8.0 ± 0.5
8.0 ± 0.5
8.0 ± 0.5
8.0 ± 0.5
Day 1
9.1 ± 0.3
12.0 ± 0.5
21.3 ± 0.3
23.3 ± 0.1
Day 2
9.8 ± 0.2
22.8 ± 0.8
25.0 ± 0.2
30.0 ± 1.6
Day 4
10.9 ± 0.2
31.9 ± 0.8
34.9 ± 0.7
43.7 ± 1.0
Day 5
10.6 ± 0.4
62.0 ± 1.0
38.4 ± 0.4
58.3 ± 0.4
Day 10
11.7 ± 0.8
60.4 ± 0.16
39.0 ± 0.2
59.0 ± 0.4
Day 15
12.7 ± 0.2
55.0 ± 1.7
38.9 ± 0.3
61.1 ± 0.6
Note: Ten-day-old seedlings (Day 0) rice seedlings were inoculated with P. indica, exposed to salt treatment, or both or no treatment for up to 15 d. The proline content was determinate at the given days. Each data represents the means of 3 independent experiments ± SE. All the data are significantly different at P < 0.05.
(10000 rpm for 10 min). The supernatant was reacted with 2 ml acid ninhydrin and 2 ml of glacial acetic acid in a capped test tube for 1 h at 100°C and the reaction terminated in an ice bath. The reaction mixture was extracted with 4 ml ice-cold toluene and mixed vigorously with a test tube glass stirrer for 15–20 s. The chromophore containing toluene was extracted from the aqueous phase, warmed to room temperature, and the absorbance was read at 520 nm using toluene as blank. Proline concentration was determined from a standard curve and calculated on a fresh weight basis as follows: [(µg proline / ml*ml toluene) /115.5 µg/µmole] /(g sample /5) = µmole proline / g of fresh weight material.34 Statistical analysis For the analyses, SigmaPlot 11.0 version was used. Paired t-test was used to check the significance difference between non-inoculated and P. indica-inoculated plants as well as when the salt treatment was given in both the conditions. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
Johri AK and Dua M are thankful to the JNU for providing DST-PURSE and capacity building grant. Jogawat A, Bakshi M, Dayaman V, MK and Saha S are very thankful to CSIR, UGC, ICMR, and DST, Government of India for granting research fellowship.
Pham GH, Kumari R, Singh A, Sachdev M, Prasad R, Kaldorf M, Buscot F, Oelmüller R, Peskan T, Weiss M, Hampp R, Varma A. Axenic cultures of Piriformospora indica. In: Varma A, Abbott L, Werner D, Hampp R, eds. Plant Surface Microbiology. Springer-Verlag 2004; 593-616. Sahay NS, Varma A. Piriformospora indica: a new biological hardening tool for micropropagated plants. FEMS Microbiol Lett 1999; 181:297302; PMID:10585552; http://dx.doi. org/10.1111/j.1574-6968.1999.tb08858.x
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5.
6.
Shahollari B, Varma A, Oelmüller R. Expression of a receptor kinase in Arabidopsis roots is stimulated by the basidiomycete Piriformospora indica and the protein accumulates in Triton X-100 insoluble plasma membrane microdomains. J Plant Physiol 2005; 162:945-58; PMID:16146321; http://dx.doi. org/10.1016/j.jplph.2004.08.012 Shahollari B, Vadassery J, Varma A, Oelmüller R. A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana. Plant J 2007; 50:1-13; PMID:17397506; http://dx.doi. org/10.1111/j.1365-313X.2007.03028.x
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7.
8.
9.
10.
11.
12.
13.
14.
Sherameti I, Shahollari B, Venus Y, Altschmied L, Varma A, Oelmüller R. The endophytic fungus Piriformospora indica stimulates the expression of nitrate reductase and the starch-degrading enzyme glucan-water dikinase in tobacco and Arabidopsis roots through a homeodomain transcription factor that binds to a conserved motif in their promoters. J Biol Chem 2005; 280:26241-7; PMID:15710607; http://dx.doi.org/10.1074/jbc.M500447200 Varma A, Savita Verma, Sudha, Sahay N, Butehorn B, Franken P. Piriformospora indica, a cultivable plantgrowth-promoting root endophyte. Appl Environ Microbiol 1999; 65:2741-4; PMID:10347070 Varma A, Singh A, Sudha Sahay N, Sharma J, Roy A, Kumari M, Rana D, Thakran S, Deka D, Bharti K, et al. Piriformospora indica: a cultivable mycorrhiza-like endosymbiotic fungus. In: Hock B, eds. Mycota IX. Germany: Springer Series, Berlin. New York: Heidelberg, 2001, 123-150. Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fischer M, Heier T, Hückelhoven R, Neumann C, von Wettstein D, et al. The endophytic fungus Piriformospora indica reprograms barley to saltstress tolerance, disease resistance, and higher yield. Proc Natl Acad Sci U S A 2005; 102:1338691; PMID:16174735; http://dx.doi.org/10.1073/ pnas.0504423102 Sun C, Johnson JM, Cai D, Sherameti I, Oelmüller R, Lou B. Piriformospora indica confers drought tolerance in Chinese cabbage leaves by stimulating antioxidant enzymes, the expression of droughtrelated genes and the plastid-localized CAS protein. J Plant Physiol 2010; 167:1009-17; PMID:20471134; http://dx.doi.org/10.1016/j.jplph.2010.02.013 Aschheim K. Research highlights: Plant probiotic. Nat Biotechnol 2005; 23:1241; http://dx.doi. org/10.1038/nbt1005-1241 Stein E, Molitor A, Kogel KH, Waller F. Systemic resistance in Arabidopsis conferred by the mycorrhizal fungus Piriformospora indica requires jasmonic acid signaling and the cytoplasmic function of NPR1. Plant Cell Physiol 2008; 49:1747-51; PMID:18842596; http://dx.doi.org/10.1093/pcp/ pcn147 Baltruschat H, Fodor J, Harrach BD, Niemczyk E, Barna B, Gullner G, Janeczko A, Kogel KH, Schäfer P, Schwarczinger I, et al. Salt tolerance of barley induced by the root endophyte Piriformospora indica is associated with a strong increase in antioxidants. New Phytol 2008; 180:501-10; PMID:18681935; http://dx.doi. org/10.1111/j.1469-8137.2008.02583.x
15. Lutts S, Majerus V, Kinet JM. NaCl effects on proline metabolism in rice (Oryza sativa L.) seedling. Physiol Plant 1999; 105:450-8; http:// dx.doi.org/10.1034/j.1399-3054.1999.105309.x 16. Jouyban Z. The effects of salt stress on plant growth. Tech J Engin & App Sci 2012; 2:7-10 17. Kumar M, Yadav V, Kumar H, Sharma R, Singh A, Tuteja N, Johri AK. Piriformospora indica enhances plant growth by transferring phosphate. Plant Signal Behav 2011; 6:723-5; PMID:21502815; http:// dx.doi.org/10.4161/psb.6.5.15106 18. Jamil M, Lee KB, Jung KY, Lee DB, Han MS, Rha ES. Salt stress inhibits germination and early seedling growth in cabbage (Brassica oleracea capitata L.). Pak J Biol Sci 2007; 10:910-4; PMID:19069887; http:// dx.doi.org/10.3923/pjbs.2007.910.914 19. Ashraf M, Sultana R. Combination effect of NaCl salinity and N-form on mineral composition of sunflower plants. Biol Plant 2000; 43:615-9; http:// dx.doi.org/10.1023/A:1002860202032 20. Akram NA, Ashraf M. Improvement in growth, chlorophyll pigments and photosynthetic performance in salt-stressed plants of sunflower (Helianthus annuus L.) by foliar application of 5-aminolevulinic acid. Agrochimica 2011; 55:94-104 21. Arfan M, Athar HR, Ashraf M. Does exogenous application of salicylic acid through the rooting medium modulate growth and photosynthetic capacity in two differently adapted spring wheat cultivars under salt stress? J Plant Physiol 2007; 164:685-94; PMID:16884826; http://dx.doi. org/10.1016/j.jplph.2006.05.010 22. Perveen S, Shahbaz M, Ashraf M. Regulation in gas exchange and quantum yield of photosystem II (PSII) in salt stressed and non-stressed wheat plants raised from seed treated with triacontanol. Pak J Bot 2010; 42:3073-81 23. Ashraf M. Harris PJC. Photosynthesis under stressful environments: An overview. Photosynthetica 2013; 51:163-90; http://dx.doi.org/10.1007/ s11099-013-0021-6 24. Gomathi R, Rakkiyapan P. Comparative lipid peroxidation, leaf membrane thermostability, and antioxidant system in four sugarcane genotypes differing in salt tolerance. Int J. Plant Physiol Biochem 2011; 3:67-74
25. Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A. Role of proline under changing environments: a review. Plant Signal Behav 2012; 7:1456-66; PMID:22951402; http://dx.doi. org/10.4161/psb.21949 26. Al-Karaki GN, Hammad R, Rusan M. Response of two cultivars differing in salt tolerance to inoculation with mycorrhizal fungi under salt stress. Mycorrhiza 2001; 11:43-7; http://dx.doi.org/10.1007/s005720100098 27. Käfer E. Mitotic crossing over and nondisjunction in translocation heterozygotes of Aspergillus. Genetics 1976; 82:605-27; PMID:773747 28. Kumar M, Yadav V, Tuteja N, Johri AK. Antioxidant enzyme activities in maize plants colonized with Piriformospora indica. Microbiology 2009; 155:78090; PMID:19246749; http://dx.doi.org/10.1099/ mic.0.019869-0 29. Dickson S, Smith SM. Evaluation of vesiculararbuscular mycorrhizal colonization by staining. In: Varma A, eds. Mycorrhiza Manual. Berlin, Germany: Springer-Verlag, 1998: 77-84. 30. Phillips JM, Hayman DS. Improved procedures for clearing roots and staining parasitic and VAM fungi for rapid assessment of infection. Trans Br Mycol Soc 1970; 55:158-61; http://dx.doi.org/10.1016/ S0007-1536(70)80110-3 31. McGonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA. A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol 1990; 15:495501; http://dx.doi.org/10.1111/j.1469-8137.1990. tb00476.x 32. Rodriguez RJ, Henson J, Van Volkenburgh E, Hoy M, Wright L, Beckwith F, Kim YO, Redman RS. Stress tolerance in plants via habitat-adapted symbiosis. ISME J 2008; 2:404-16; PMID:18256707; http:// dx.doi.org/10.1038/ismej.2007.106 33. Mane AV, Karadge BA, Samant JS. Salinity induced changes in photosynthetic pigments and polyphenols of Cymbopogon nardus (L.) Rendle. J Chem Pharm Res 2010; 2:338-47 34. Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies. Plant Soil 1973; 39:205-7; http://dx.doi.org/10.1007/BF00018060
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