http://dx.doi.org/10.5423/PPJ.OA.11.2011.0210 pISSN 1598-2254 eISSN 2093-9280
The Plant Pathology Journal
© The Korean Society of Plant Pathology
Induced Systemic Drought and Salt Tolerance by Pseudomonas chlororaphis O6 Root Colonization is Mediated by ABA-independent Stomatal Closure
Song Mi Cho1†, Beom Ryong Kang2†, Jeong Jun Kim3 and Young Cheol Kim4*
1Department of Floriculture, Chunnam Techno College, Gokseong 516-911, Korea
2Environment-Friendly Agricultural Research Institute, Jeollanamdo Agricultural Research and Extension Services, Naju 520- 715, Korea
3Agricultural Microbiology Team, National Academy of Agricultural Science, Rural Development Administration, Suwon 441- 707, Korea
4Institute of Environmentally-Friendly Agriculture, Department of Plant Biotechnology, Chonnam National University, Gwangju 500-757, Korea
(Received on November 13, 2011; Revised on January 5, 2012; Accepted on January 11, 2012)
Root colonization by the rhizobacterium Pseudomonas chlororaphis O6 in Arabidopsis thaliana Col-0 plants resulted in induced tolerance to drought and salinity caused by halide salt-generated ionic stress but not by osmotic stress caused by sorbitol. Stomatal apertures decreased following root colonization by P. chlororaphis O6 in both wild-type and ABA-insensitive Arabidopsis mutant plants. These results suggest that an ABA- independent stomatal closure mechanism in the guard cells of P. chlororaphis O6-colonized plants could be a key phenotype for induced systemic tolerance to drought and salt stress.
Keywords : abiotic stress, induced tolerance to environ- mental stress, priming, stomatal closure
Plants possess various survival systems against abiotic stresses, such as cold, drought, and salinity (Zhu, 2001).
Under environmental stresses, the level of the plant hormone, abscisic acid (ABA) increases triggering adaptive responses essential for survival (Zhu, 2001). During drought and salt stresses, ABA induces stomatal closure to minimize water loss through transpiration (Leung and Giraudat, 1998). Consequently, ABA-biosynthesis mutants (aba mutants) and some of the ABA-response plant mutants (i.e. the ABA-insensitive abi mutants) are susceptible to drought stress, due to problem of stomatal aperture regulation (Leung and Giraudat, 1998; Schroeder et al., 2001). However, another plant growth regulator, jasmonate (Evans, 2003; Suhita et al., 2004), also stimu- lates stomatal closure under drought conditions (Creelman
and Mullet, 1997).
Root colonization of certain plant-associated microbes elicits physiological and biochemical change to enhance systemic resistance against various biotic and abiotic stresses in plants (Kim et al., 2011; Yang et al., 2010), termed to as “induced systemic resistance (ISR)” or
“induced systemic tolerance (IST)”. IST against drought or salt stresses in plants can be induced with systemic application of certain rhizobacteria including Gram- positive Bacillus strains (Ryu et al., 2004; Timmusk and Wagner 1999; Zhang et al., 2010), an endophytic fungal isolate, Trichoderma harziarum (Bae et al., 2010), and certain Gram-negative bacterial isolates, such as ACC- deaminase producing bacteria (Mayak et al., 2004). Root colonization by Pseudomonas chlororaphis O6 induced systemic resistance against a broad spectrum of plant diseases caused by viral, bacterial, and fungal pathogens in various plants by jasmonic acid-ethylene related pathways (Cho et al., 2008; Kim et al., 2004; Ryu et al., 2007;
Spencer et al., 2003). Additionally, root colonization by P.
chlororaphis O6 induced systemic tolerance against drought, a process accompanied by stomatal closure. Applying 2R,3R-butanediol, a volatile produced by P. chlororaphis O6, resulted in induced tolerance through a salicylic acid (SA), jasmonic acid (JA) and ethylene-dependent mech- anism (Cho et al., 2008). However, mechanisms involved in microbe-mediated IST against drought have not been characterized or elucidated.
In this study, we tested the hypothesis that P. chloro- raphis O6 induces tolerance to other abiotic stresses, such as salinity, osmotic pressure, cold and heat. We used addi- tional Arabidopsis mutants altered in ABA signaling pathways to identify the role of ABA in induced abiotic stress tolerance. Parental A. thaliana ecotypes Columbia (Col-0) or Landsberg erecta (Ler), and mutant and trans- genic lines were obtained from the Ohio State University
*Corresponding author.
Phone) +82-62-530-2071, FAX) +82-62-530-0208 E-mail) yckimyc@chonnam.ac.kr
† These authors contributed equally to this work.
Note Open Access
Stock Center, Ohio State University (Columbus, OH, USA).
Mutant lines included abi3-1 (ABA-insensitive, acidic domain transcription factor), and abi4-1 (ABA-insensitive, APETALA2 domain transcription factor) (the Salk Ins- titute, La Jolla, CA, USA), A333 and A313 (over-express- ing the ABA-response factor ABF3) and AK313 (an ABF3 knock out mutant) were used (Kang et al., 2002).
Arabidopsis plants were grown on agar plates described previously (Cho et al., 2008). Briefly, the surface-steriliz- ed seeds were placed singly into the wells of microtiter plates (12-well microtiter, SPL inc., Seoul, South Korea) containing half-strength Murashige and Skoog salt (MS) medium, 0.3% phytagel and 3% sucrose, adjusted to pH 5.7. Other washed sterile seeds were added singly onto the surface of sterile Whatman #1 filter paper and placed over the half strength MS medium contained in the microtiter wells. The microplates were sealed with Parafilm to pre- vent drying, and no additional water was applied during the experiment.
P. chlororaphis O6 (Spencer et al., 2003) and Pseudo- monas fluorescens 89B61 (Ryu et al., 2004) were used as ISR-inducing bacterial strains, and an Escherichia coli DH5α was used as a negative control. All bacteria were streaked onto King’s medium B agar plates and incubated at 28 °C for 2 days prior to experiments. The bacteria were grown overnight in KB broth, pelleted by centrifugation, washed once with sterile water, and suspended to 1× 108 colony-forming unit (cfu)/ml.
Two week-old Arabidopsis seedlings grown in microtiter plates were treated on roots with 10–15 µl of inoculum [(1× 108 cfu/ml)/plant] of P. chlororaphis O6, P. fluore- scens 89B61, E. coli DH5α suspension, or water as a control. One week after the bacterial root treatments, the microplate-grown seedlings were transferred on their filter paper to one-half-strength MS medium supplemented with 200 mM NaCl, 200 mM KCl, or 200 mM sorbitol to assess salt and osmotic resistance. The number of dead plants in each treatment was counted 5−7 days after exposure to the salt stresses.
P. chlororaphis O6 root colonization increased the sur- vival of plants exposed to 200 mM NaCl or KCl (Fig. 1A).
About 90% plants were survived in P. chlororaphis O6- colonized plants, but only about 20% of the plants for the controls. Plants inoculated with P. chlororaphis O6 showed a significantly higher survival rate when the growth medium was amended with 200 mM NaCl. However, when the plants were treated with 200 mM sorbitol to generate osmotic stress, no protection was observed (Fig. 1B). However, other ISR-eliciting rhizobacteria, such as P. fluorescens 89B61 and E. coli DH5α as a negative control did not show systemic salt tolerance (Fig. 1B). These results indicated that P. chlororaphis O6-treated plants at the young seedling
stage resulted in induction of tolerance to salinity but not to all increases in osmotic pressure.
We explored whether root colonization of P. chlororaphis O6 could induce systemic tolerance to drought in A.
thaliana plants grown in microtiter plates. Seven days after bacterial treatment, leaves of plants were excised, and then weight of each leaf was measured every 20 min over a period of 2 h (Kang et al., 2002). Assays were performed three times using ten plants per treatment. Less weight loss was observed in leaves from P. chlororaphis O6-colonized plants than leaves from non-colonized plants, or those colonized by other bacteria. Two hours after drought treatment, P. chlororaphis O6 treated plant leaves had lost only 59% of their weight, whereas leaves from plants without inoculation had lost 80−90% of the original weight (Fig. 2). In P. fluorescens 89B61 or E. coli DH5α treated plants, relative weight losses were significantly higher than that of the water-treated plants (Fig. 2). We do not know exact mechanism why more weight losses occurred in the plants treated with 89B61 and DH5α, but we speculate that certain microbes might have a negative effect under Fig. 1. Effect of 200 mM NaCl, KCl, and sorbitol on Arabidopsis plants grown with and without bacterial root inoculations.
Seedlings of Col-0 plants were grown for 2 weeks in half strength MS-medium, before treatment with Pseudomonas chlororaphis O6 (O6), P. fluorescens 89B61 (89B61), Escherichia coli DH5α (Ecoli), or water as a control. One week after bacterial treatment, the plants were transferred to MS medium with or without NaCl, KCl and sorbitol amendments. (A) Photo-images were taken of plants after exposure to NaCl or KCl. (B) Data shown are the survival percentages for the abiotic stressed plants relative to the survival of non-exposed plants. Three independent experiments were performed with at least 12 plants per treatment. Different letters indicate significant differences between treated and control samples based on Duncan’s multiple range test at P < 0.05.
drought or salt stresses. These results indicated that systemic drought and salt tolerance induced by P. chlororaphis O6 might be the result of reduced transpiration in the leaves of Arabidopsis plants with roots colonized by P. chlororaphis O6.
To investigate the signaling pathways involved in stomatal closure in the P. chlororaphis O6-colonized plants, we used abi3 and abi4 mutants, which are ABA insensitive. A.
thaliana plants were grown in the microtiter plates for two weeks, and roots were treated with bacteria or water as a control for five days. The leaves were stained with Safranin O (0.5%, Sigma, St. Louis, MO, USA) for 0.5−1 min and washed three times with sterile distilled water to examine stomatal aperture size. Samples were observed under light microscopy (Zeiss, Model DE/AXIOLAB-POI, Gena, Germany) and obtained images (Image-pro plus 4.5.1, Media Cybernetics Inc., Silver Spring, MD, USA). Stomatal aperture size was measured using an image acquisition and process- ing system program (Focus, Focus Com., Daejeon, South Korea). At least 100 stomata were counted on five plants per treatment.
Stomatal apertures of wild-type Col-0 were about 4.8 µm for the leaves of plants grown without P. chlororaphis O6 colonization, whereas those for the ABA insensitive lines Fig. 2. Relative plant weight loss of Arabidopsis roots treated
with the different bacterial strains. Plant weight was recorded at 20 minute intervals using leaves from Col-0 plants grown in half- strength MS medium and treated with Pseudomonas chlororaphis O6 (O6), P. fluorescens 89B61 (89B61), Escherichia coli DH5α (DH5α), or water. Each data point represents the mean of three replicate experiments; standard deviations are shown as bars.
Three independent experiments were performed with 10 plants per treatment. Different letters indicate significant differences between treated and control samples based on Duncan’s multiple range test at P < 0.05.
Fig. 3. Effect of Pseudomonas chlororaphis O6 root colonization on stomatal apertures in Arabidopsis thaliana lines with altered abscisic acid (ABA) signaling pathway. A. thaliana seedlings were grown for 2 weeks in half strength MS medium, before root treatment with P. chlororaphis O6 (O6) or water as a control. Five days after treatment, photo-images were taken from A. thaliana plants under a microscope (A). Stomatal apertures were measured from A. thalina ABA-insensitive lines that were grown with or without root inoculation of P. chlororaphis O6 (B) or water-treated ABF3 deficient (AK313) and ABF3 overexpressed mutant (A333) lines (C). At least 100 stomata per control treatment were observed under a microscope in three separate trials. Each data point represents the mean of three replicate experiments, and different letters indicate significant differences between treated and control samples based on Duncan’s multiple range test at P < 0.05.
abi3 and abi4 were approximately 4 µm (Fig. 3A, 3B).
Mutant line AK313, which is ABF3 deficient, also had apertures of about 4.0 µm. Apertures for the A333 mutant, which overexpresses the ABA-response factor ABF3, were about 2.5 µm in non-inoculated plants. Stomatal apertures in the P. chlororaphis O6-inoculated wild type and mutant abi-insensitive plants were similar at about 3 µm (Fig. 3C).
Thus, the bacterium caused stomatal closure in Arabidopsis lines that were not responsive to ABA.
In this study, we showed that reduced water loss in P.
chlororaphis O6-colonized plants correlated with smaller stomatal aperture. Whether ABA is required for stomatal closure in IST awaits further clarification. One possible mechanism for bacteria-mediated IST against drought is a priming effect upon root colonization with P. chlororahis O6 before drought stress. The decrease in stomatal aperture was detectable as a result of the P. chlororaphis O6 priming effect (Cho et al., 2008; 2011). In this study, the induced drought and salt resistance by P. chlororaphis O6 was accompanied by a lower transpiration level than that in non- colonized plants, which correlated with the reduced stomatal aperture. The decrease in stomatal aperture was detectable 3 days after root colonization (Cho et al., 2008).
In addition, induction of tolerance to abiotic stress by P.
chlororaphis O6 root colonization was limited to drought and salt stress. P. chlororaphis O6 root-colonized Arabi- dopsis plants did not show induced systemic tolerance against heat, cold or freezing (data not shown).
Previous studies showed that plant defense signal path- ways are important to induce stomatal closure by microbes and microbe-associated molecular patterns-mediated IST (Cho et al., 2008; 2011; Melotto et al., 2006). P. chlororaphis O6 requires the SA, JA and ethylene pathways instead of ABA accumulation, as described in the general mechanism of drought tolerance (Cho et al., 2008). Evidence of a role for SA-signaling rather than ABA-signaling in IST by microbes and pathogen-associated molecular patterns was shown in other studies reporting that SA causes stomatal closure and tolerance to drought and salinity (Bezrukova et al., 2001). Whether ABA is required for stomatal closure in IST awaits further clarification.
The extent of closure was similar to that observed in plant lines over-expressing the ABF3 ABA-response factor (Kang et al., 2002). However, the P. chlororaphis O6-colonized plants showed none of the abnormal phenotypes manifested in the ABF3-overexpressing line such as epinasty of leaves and an abnormal obstacle-touching response (Kang et al., 2002). A recent study indicated that plant stomata closure is induced by local application of bacterial surface molecules from both plant pathogenic and nonpathogenic bacteria.
This process is viewed as a plant innate immunity response to restrict entry of plant pathogens (Melotto et al., 2006).
Our studies showed that P. chlororaphis O6 root coloni- zation also closes stomates, although we have not observed colonization of aerial tissues by this bacterium (data not shown). In both systems, stomatal closure requires the SA signaling pathway (Melotto et al., 2006).
In summary, our results indicated that induced systemic drought tolerance by P. chlororaphis O6 could be ABA- independent, although ABA-dependent abiotic stress related genes are parts of the expressed defense gene arsenal. We suggest that SA and/or JA production resulting from root colonization by P. chlororaphis was a key regulatory feature of stomata closure and resulted in induced tolerance to drought and salt stress (Cho et al., 2008). The stomatal closure displayed in P. chlororaphis O6-colonized Arabi- dopsis plants could be a major factor maintaining plant viability under drought and salt stress.
Acknowledgements
We thank Dr. J. W. Kloepper (Auburn University, AL, USA) for providing ISR-inducing strains, Dr. Kim (Chonnam National University, Gwangju, Korea) and the Arabidopsis Biological Resources Center at Ohio State University (Columbus, OH, USA) and the Salk Institute (La Jolla, CA, USA) for the mutant lines used in this research. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0011555).
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