Paenibacillus polymyxa BFKC01 enhances plant iron absorption via improved root systems and activated iron acquisition mechanisms

Plant Physiology and Biochemistry 105 (2016) 162e173

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Research article

Paenibacillus polymyxa BFKC01 enhances plant iron absorption via improved root systems and activated iron acquisition mechanisms Cheng Zhou a, 1, Jiansheng Guo b, 1, Lin Zhu c, Xin Xiao a, Yue Xie a, Jian Zhu c, Zhongyou Ma a, **, Jianfei Wang a, * a Key Laboratory of Bio-organic Fertilizer Creation, Ministry of Agriculture, Institute for Applied Microbiology, Anhui Science and Technology University, Bengbu 233100, China b School of Medicine, Zhejiang University, Hangzhou 310058, China c Department of Molecular and Cell Biology, Tongji University, Shanghai 200092, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 January 2016 Received in revised form 13 April 2016 Accepted 14 April 2016 Available online 14 April 2016

Despite the high abundance of iron (Fe) in most earth's soils, Fe is the major limiting factor for plant growth and development due to its low bioavailability. With an increasing recognition that soil microbes play important roles in plant growth, several strains of beneficial rhizobactria have been applied to improve plant nutrient absorption, biomass, and abiotic or biotic stress tolerance. In this study, we report the mechanisms of microbe-induced plant Fe assimilation, in which the plant growth promoting rhizobacteria (PGPR) Paenibacillus polymyxa BFKC01 stimulates plant's Fe acquisition machinery to enhance Fe uptake in Arabidopsis plants. Mechanistic studies show that BFKC01 transcriptionally activates the Fedeficiency-induced transcription factor 1 (FIT1), thereby up-regulating the expression of IRT1 and FRO2. Furthermore, BFKC01 has been found to induce plant systemic responses with the increased transcription of MYB72, and the biosynthetic pathways of phenolic compounds are also activated. Our data reveal that abundant phenolic compounds are detected in root exudation of the BFKC01-inoculated plants, which efficiently facilitate Fe mobility under alkaline conditions. In addition, BFKC01 can secret auxin and further improved root systems, which enhances the ability of plants to acquire Fe from soils. As a result, BFKC01-inoculated plants have more endogenous Fe and increased photosynthetic capacity under alkaline conditions as compared to control plants. Our results demonstrate the potential roles of BFKC01 in promoting Fe acquisition in plants and underline the intricate integration of microbial signaling in controlling plant Fe acquisition. © 2016 Elsevier Masson SAS. All rights reserved.

Keywords: Paenibacillus polymyxa Iron deficiency Calcareous soils Soil microbes Phenolic compounds

1. Introduction Iron (Fe) is essential for plant growth and physiological processes such as photosynthesis and respiration (Palmer and Guerinot, 2009). Despite the high abundance of Fe in most soils, its bioavailability is much lower in alkaline or calcareous soils. Fe forms very scarcely soluble Fe3þ oxy-hydroxides, which are not readily utilized by plants (Guerinot and Yi, 1994). Calcareous soils cover approximately one-third of the earth's crust, and many plant species grown in these soils often display chlorosis and yield losses

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Z. Ma), [email protected] (J. Wang). 1 Cheng Zhou and Jiansheng Guo equally contributed to this work. http://dx.doi.org/10.1016/j.plaphy.2016.04.025 0981-9428/© 2016 Elsevier Masson SAS. All rights reserved.

associated with Fe deficiency (Abadía et al., 2011). For agricultural sustainability, there is an urgent need to enhance the ability of plants to assimilate Fe from low Fe-availability soils. Plants have evolved two major strategies to acquire Fe from soils € mheld, 1994; Brumbarova et al., 2015). Non(Marschner and Ro graminaceous monocots and dicots primarily use a reductionbased strategy (strategy I). The mechanism includes three steps: (I) Release of protons into the rhizosphere decreases the rhizosphere pH and increases the solubility of Fe3þ oxy-hydroxides via the H-ATPase (AHA2) (Santi and Schmidt, 2009); (II) Ferric (Fe3þ) is reduced to the ferrous (Fe2þ) form by the plasma membrane bound ferric chelate reductase (FRO2) (Connolly et al., 2003); (III) The reduced Fe2þ is transported across plasma membrane into root epidermic cells via the divalent metal transporter IRT1. FRO2 and IRT1 are finely regulated by the Fe-deficiency-induced transcription factor (FIT1), which plays central roles in Fe homeostasis

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(Colangelo and Guerinot, 2004). Graminaceous monocots acquire Fe based on a Fe-chelating strategy (strategy II). Phytosiderophores are secreted into the rhizosphere for chelating Fe, and the resulting Fe-chelating complexes are then transported into root epidermic cells (Gendre et al., 2007). Intriguingly, non-graminaceous monocots and dicots have been reported to employ a chelation-based Fe acquisition strategy by exudation of phenolic compounds to chelate Fe (Schmid et al., 2014). Previous studies have demonstrated that the protons released by non-graminaceous plants are largely buffered by high pH or calcareous soils, and the ferric chelate reductase activities are hampered (Ohwaki and Sugahara, 1997). This indicates that the reduction-based strategy severely stalls under these conditions. Therefore, strategy I plants rely on root exudation of phenolic compounds to chelate Fe rather than the reduction-based strategy under alkaline conditions. These phenolic compounds have indeed complexing capacity for Fe for their very high affinity constant (up to logk ¼ 40), yet some also have a reducing capacity contributing to Fe mobilization mechanisms (Schmid et al., 2014). By contrast, most graminaceous plants exhibited better growth than nongraminaceous monocots and dicots under alkaline conditions (Nozoye et al., 2011). Unlike the reduction-based strategy, the phytosiderophore-dependent Fe acquisition is relatively insensitive € mheld and Marschner, 1986). However, either to high-pH soils (Ro graminaceous or nongraminaceous plants cannot obtain enough Fe in alkaline or calcareous soils by strategy I or II Fe acquisition mechanisms. Masalha and Kosegarten (2000) reported that sun flower (Helianthus annuus L.) grown in sterilized calcareous soils exhibited poor growth and typical symptoms of Fe deficiency as compared to control plants grown in non-sterilized soils. Similarly, the growth of rape was seriously inhibited in sterilized soils, but its growth quickly recovered after soil application of Feethylenediaminedihydroxyphenylacetic acid (EDDHA) (Rroco et al., 2003). Thus, soil microbes seem to play important roles in assisting plants to efficiently acquire Fe. Several strains of soil microbes have been found to colonize and flourish in the plant rhizosphere, and some of them exert beneficial effects on plant growth via diverse ways including synthesis of plant-growth-regulation factors, promotion of nutrient uptake, and control of pathogenic invasion (Dey et al., 2004; Scholz et al., 2011; Pii et al., 2015a,b). These soil bacteria are collectively referred to as plant growth promoting rhizobacteria (PGPR). Some species of rhizobacteria such as Bacillus and Paenibacillus are PGPR (Cavaglieri et al., 2005; Jin et al., 2011). Paenibacillus polymyxa, the type species of the genus Paenibacillus, is considered to be of great importance for plant growth. Several strains of P. polymyxa have been identified and are widely used in sustainable agriculture (Anand et al., 2013). In this study, we examined the activity of P. polymyxa strain BFKC01 in plant Fe assimilation. BFKC01 triggered Fe deficiency responses and significantly enhanced releases of phenolic compounds in Arabidopsis plants. Consequently, BFKC01-inoculated plants displayed notably better growth than non-inoculated plants under alkaline or calcareous soil conditions. These results indicated that BFKC01 had the potential to improve plant uptake of Fe. 2. Materials and methods 2.1. Plant materials and growth conditions Seeds of Arabidopsis thaliana (Columbia ecotype) were surface sterilized with 0.1% (w/v) HgCl2 for 5 min and then rinsed three times with sterile water. The sterilized seeds were cultured on 1/2 MS agar medium with 50 mM Fe(III)-EDTA and placed in growth cabinets at 21  C with a 14 h/10 h light/dark cycle. After 12 days (d)

163

of growth, the seedlings were transferred into MS agar plates (pH ¼ 7.2) with different concentrations of Fe(III)-EDTA (severe Fe limitation, 5 mM; mild Fe limitation, 20 mM; sufficient Fe, 50 mM) or alkaline soils, and were subjected to different treatments, depending on the experiments. The pH of MS agar medium was adjusted to 7.2 by addition of KOH. In addition, alkaline soils were prepared according to the method reported by Kim et al. (2006). Calcium oxide (CaO) (Sigma, 3 g or 6 g CaO/kg dry soil) was added to the soils (3:1:1, Pro-Mix: vermiculite: perlite) to generate alkaline soils. Then, dry soils were diluted with distilled water at the ratio of 1:1 (w/v), and these samples were used to measure pH accreting to the method reported by Thunjai et al. (2007). The pH of soils supplemented with 0.3% or 0.6% (w/w) CaO is approximately 7.4 or 7.8, respectively. Lastly, the non-calcareous or calcareous soils were sterilized by autoclaving at 120  C for 1 h according to the method described by Masalha and Kosegarten (2000) with minor modifications, and the soils were incubated before using it. 2.2. Bacterial isolation, culture and inoculation P. polymyxa strain BFKC01 was isolated from cultivated maize plants and was stored at 80  C in sterile water with 20% (v/v) glycerol. BFKC01 was confirmed by 16S rDNA sequencing (Genbank no. KT887958). At 1 d before bacterial inoculation experiments, BFKC01 was initially grown in tryptic soy agar plates at 30  C. The bacteria was then collected from the plates in deionized water to yield 109 colony forming units (CFU) ml1, according to the method as described by Zhang et al. (2009). 5 ml of bacterial suspension culture was inoculated and evenly distributed into roots of 12-dayold Arabidopsis seedlings, and these plants were then grown vertically on MS agar plates. To further evaluate the effects of BFKC01 on Arabidopsis plants grown in calcareous soils, 12-day-old seedlings were transferred from MS agar plates into sterilized soils, then 1 ml of bacteria suspension (103 CFU ml1) was inoculated into the roots of each plant. 2.3. Analyses of chlorophyll content and photosynthetic efficiency To determine chlorophyll content, about 500 mg of leaf samples was homogenized in 5 ml of 80% (v/v) aqueous acetone and then centrifuged at 12,000 g for 15 min. Absorbance of the supernatant was measured at 645 and 663 nm, respectively. Total chlorophyll content was calculated using the formula (20.21  A645 þ 8.02  A663) described by Porra (2002). The photosynthetic rate (Pn) was measured at 21  C and 60% relative humidity with a portable CIRAS-2 system. Photon flow density was controlled by the automatic control function of the CIRAS-2 system. Chlorophyll fluorescence was measured according to a recently described method (Du et al., 2015). The ratio Fv/Fm ¼ (FmeFo)/Fm was used to assess maximal efficiency of the PSII, and FPSII ¼ (Fm0 eF)/Fm0 was used to estimate actual efficiency of the PSII. 2.4. Determination of Fe and IAA concentration The Fe concentration in plant tissues was examined using the method of Lobreaux and Briat (1991). Samples were extracted from whole plants and rinsed five times with deionized water before measuring Fe concentration. About 500 mg of plant tissue was ground in liquid N2 and was then mineralized by sequential treatment with HNO3, H2SO4, and HClO4 as described by Beinert (1978). The Fe concentration was analyzed by measuring absorbance of the Fe2þ-phenanthroline complex at 510 nm using thioglycolicacid as the reducing agent. Microbial production of IAA in the culture medium was determined as described recently by Mei et al. (2014). Endogenous IAA in plant tissues was analyzed by gas

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chromatography using the method reported by Ljung et al. (2005).

3. Results

2.5. Transmission electron microscopy

3.1. BFKC01 improves plant growth under Fe deficiency

Leaf samples were cut into 0.5  0.5 cm pieces, and were immediately fixed in 2.5% glutaraldehyde at 23  C for 4 h. After three rinses with PBS (0.1 M, pH 7.2), the samples were fixed with 1.0% osmium tetroxide (OsO4) for 2 h, followed by three rinses with PBS. Samples were then dehydrated in an acetone dilution series from 50 to 100%, embedded in Eponate resin 12 (Ted Pella; USA), and cut into thin sections (70 nm). The ultrathin sections were observed by transmission electron microscopy (TEM). At least three seedlings and nine individual chloroplasts were observed. Each sample was sectioned 3 times and typical photographs were selected.

Distribution of IAA was examined using DR5::GUS Arabidopsis seedling. The non-inoculated or bacteria-inoculated seedlings were immersed in aqueous acetone (90%, v/v) for 20 min and then transferred into the GUS staining solution 2 mM 5-bromo-4chloro-3-indolylglucuronide (X-Gluc), 50 mM sodium phosphate (pH 7.2), 2 mM potassium ferricyanide, 2 mM potassium ferrocyanide and 0.2% (v/v) Triton X-100 for 18 h at 37  C. The seedlings were then bleached with 70% ethanol and observed using a Nikon Eclipse 80i microscope.

To assess whether BFKC01 increased the plant ability to absorb Fe under alkaline conditions, the Arabidopsis seedlings were initially cultured on MS agar medium for 12 d and were then transferred to MS agar medium (pH ¼ 7.2) or calcareous soils with or without exposure to BFKC01, respectively. As shown in Fig. 1A, the BFKC01-inoculated plants exhibited better growth on MS agar plates containing 5 mM, 20 mM or 50 mM Fe(III)-EDTA, respectively. At 8 days post inoculation (dpi), these plants displayed more lateral roots and plant biomass, and shorter primary root than noninoculated (control) plants (Fig. 1B and C). At 5 mM Fe(III)-EDTA, the control plants displayed serious symptoms of Fe deficiency including inhibited growth and leaf chlorosis, while the chlorotic symptoms were significantly less in the BFKC01-inoculated plants. We investigated the effects of BFKC01 on Arabidopsis plants grown in calcareous soils (Fig. 1D). Calcium oxide (CaO) was added to soils to produce alkaline conditions, and the high pH in calcareous soils led to a significant reduction in Fe availability. Similar to the observation on alkaline medium, leaves of non-bacteria inoculated (control) plants were chlorotic when plants were grown in soils containing 0.3% (w/w) CaO (pH ¼ 7.4), and plant growth was markedly reduced in soils (pH ¼ 7.8) containing 0.6% (w/w) CaO. However, BFKC01-inoculated plants exhibited better growth phenotypes as compared to the control plants.

2.7. Gene expression analyses

3.2. BFKC01 increases endogenous Fe concentration of plants

Total RNA was isolated from plant tissues using TRIzol (Invitrogen, USA). First-strand cDNA was synthesized from 500 ng of total RNA by reverse transcriptase ReverTra Ace kit (Takara, Japan) as the templates of reverse transcription (RT)-PCR or quantative PCR (qPCR) analyses. The PCR parameters for RT-PCR analyses were as follows: 94  C (5 min); 26 cycles of 95  C (30 s), 55  C (30 s), 72  C (30 s), and 72  C (7 min). Moreover, qPCR reactions were performed on an ABI Model 7500 using the following conditions: 30 s at 94  C; 15 s at 95  C, 30 s at 60  C for 40 cycles. All reactions were conducted in triplicate for each sample with the SYBR Green qPCR kit (Takara, Japan), and relative transcript levels of targeted genes were normalized to the Arabidopsis ACTIN2 mRNA. The primers used in above experiments are listed in supplementary Table1.

Inoculation with BFKC01 mitigated Fe deficiency-induced leaf chlorosis, suggesting that BFKC01 promoted Fe absorption of plants under alkaline or calcareous soil conditions. Levels of endogenous Fe were measured in both the control and BFKC01-inoculated plants grown at different concentrations of Fe(III)-EDTA (Table 1). When plants were grown at 5 mM Fe-EDTA, the BFKC01-inoculated plants accumulated more Fe than the control plants after 8 dpi. The Fe concentration of plants grown at 20 mM Fe-EDTA was more than 2-fold than that in the control plants. The BFKC01-inoculated plants had over 3-fold increase in the concentration of Fe as compared to the control plants when plants were grown at 50 mM Fe(III)-EDTA. Similar results were observed for the BFKC01-inoculated plants which had greater Fe accumulation than the control plants under calcareous soil conditions, with 8 d of BFKC01 treatment (Table 1).

2.6. GUS staining

2.8. Detection of ferric reductase activity and fluorescent phenolic compounds Root-associated ferric reductase activity was determined according to the method described by Zhang et al. (2009). Fluorescent phenolic compounds secreted by plant roots were monitored in the MS agar plates under UV light (365 nm). Quantification of fluorescent phenolic compounds was performed using a 96-well microplate-based colorimetric method according to the method described by Zamioudis et al. (2014). Fluorescence emitted by root exudates (excitation at 360 nm; emission at 528 nm) was detected with a Synergy Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) after removing plants and/or bacteria from the medium. 2.9. Statistical analyses Each experiment was performed three times. The mean values ± SE of at least three replicates are indicated, and the asterisks above the bars denote significant differences between the control and BFKC01-inoculated plants using a Student t-test (*p < 0.05).

3.3. BFKC01 produces IAA and promotes lateral root initiation To determine if BFKC01 could produce and secrete IAA, the culture supernatant of BFKC01 was examined for IAA. The concentration of IAA produced by BFKC01 was measured throughout bacterial growth in nutrition broth supplemented with tryptophan (Trp). As shown in Fig. 2A, low concentration of IAA was observed in the culture supernatant of BFKC01 during exponential growth, followed by a remarkable increase in the concentration of IAA at 16 h (beginning of stationary phase). However, the concentration of IAA also increased slightly during the stationary phase of bacterial growth. We found that the levels of IAA were clearly altered in the BFKC01-inoculated plants. With the time-delay of BFKC01 treatment, the concentration of IAA in the BFKC01-inoculated plants displayed an increasing tendency, but this did not occur in the control plants (Fig. 2B). Furthermore, 12-day-old DR5:GUS Arabidopsis seedlings were used to monitor the distribution of IAA. As shown in Fig. 3AeF, BFKC01-inoculated seedlings showed strong DR5:GUS staining signal, which occurred in the differentiated

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Fig. 1. The BFKC01-inoculated Arabidopsis plants exhibited better growth traits in alkaline or calcareous soils as compared to non-inoculated (control) plants. (A) The control or BFKC01-incoculated seedlings were grown on MS agar plates with different concentrations of Fe(III)-EDTA for 8 days. (B) Primary root length and (C) lateral root density of the control and BFKC01-inoculated plants. (D) The non-inoculated or BFKC01-incoculated seedlings were grown on soils supplemented with different percent (w/w) of CaO for 8 days. Data were shown as the means ± SE of at least three replicates, and the bars with asterisks indicated significant differences between the control and BFKC01-incoulated plants using a Student's t-test at P < 0.05.

Table 1 Iron concentration of the non-inoculated (control) and BFKC01-inoculated Arabidopsis plants grown under alkaline conditions. Data were shown as the means ± SE of at least three replicates, and the asterisks significant differences between control and BFKC01-incoulated plants using a Student's t-test at P < 0.05. Fe(III)-EDTA

Control BFKC01 CaO (w/w) Control BFKC01

5 mM

20 mM P < 0.05

Mean

SE

45.4 85.7 0.0% 168.2 208.6

3.6 6.9

*

12.6 15.3

*

50 mM P < 0.05

Mean

SE

138.6 180.5 0.3% 68.6 132.5

7.2 15.6

*

3.8 8.1

*

zones, vascular parenchyma of root tissues and the base of lateral roots after 48 h of bacterial inoculation. However, a weak DR5:GUS signal was only observed in the vascular parenchyma and root tips of control plants. Ultrastructure observing also revealed that cell division was rapidly induced in the vascular tissues of the BFKC01treated roots (Fig. 3G), but only fewer cells divided in the vascular tissues of the control plants (Fig. 3H).

Mean

SE

P < 0.05

195.8 258.7 0.6% 28.4 91.7

9.7 17.5

*

2.3 6.5

*

observed in the BFKC01-inoculated plants grown in soils with 0.3% CaO, but it was rarely seen in the control plants (Fig. 4B and E). The BFKC01-inoculated plants also exhibited more complete development of grana lamellae than the control plants when plants were grown in soils with 0.6% CaO (Fig. 4C and F).

3.5. Effects of BFKC01 on chlorophyll content and photosynthetic efficiency

3.4. Effects of BFKC01 on chloroplast ultrastructure in plants Fe deficiency affects the structure and functions of chloroplast in plants (Chen et al., 2015). In this study, the Arabidopsis plants had chlorotic leaves under alkaline conditions, indicating disturbance of chloroplast development. Ultrastructure analyses showed that chloroplasts in mesophyll cells from both the control and BFKC01treated plants were fully developed in non-calcareous soils (Fig. 4A and D). There were no differences in the numbers of normal grana stacking and grana lamellae between the control and BFKC01inoculated plants. When plants were grown in calcareous soils, more rudimentary grana lamellae occurred in the plastids, displaying classic traits of thylakoid disorganization induced by Fe deficiency. In contrast, the chloroplasts in mesophyll cells from the treated plants had more normal grana stacking than the control plants. For instance, plenty of normal grana stacking were clearly

Calcareous soils often lead to significant reduction of Fe availability affecting plant photosynthetic capacity (Sivitz et al., 2012). Thus, we assessed whether BFKC01 improved the photosynthesis of plants grown in calcareous soils. 12-day-old Arabidopsis seedlings grown on MS agar plates were transferred into the soils containing 0.0%, 0.3% and 0.6% CaO, respectively. These plant roots were then inoculated with BFKC01. After 8 dpi, the chlorophyll content was significantly higher in the BFKC01-inoculated plants than the control plants grown in calcareous soils, but there were no significant differences between the control and BFKC01-inoculated plants grown in non-calcareous soils (Fig. 5A). Furthermore, several photosynthetic parameters were analyzed in the control and BFKC01-inoculated plants. Light-response curves showed that Pn was greater in the BFKC01-inoculated plants than in the control plants (Fig. 5B). Previous studies have indicated that

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A IAA OD600

7

1.8 1.5

3.7. BFKC01 stimulates plant ISR with activated the transcription of MYB72 in plants

6 1.2

5 4

0.9

3

OD600

IAA concentration (μg ml-1)

8

compared to the controls (Fig. 6B and C). Ferric reductase activity was also significantly higher in the BFKC01-inoculated plants than the controls (Fig. 6D and E).

0.6

2 0.3

1

0

0 0 h 4 h 8 h 12 h 16 h 20 h 24 h 28 h 32 h 36 h 40 h

B

IAA concentration (ng g-1)

30 25

Control BFKC01

* *

20 15 10 5

In plants, induced systemic resistance (ISR) is stimulated by certain soil-borne microbes (Lee et al., 2012; Zamioudis et al., 2014). Importantly, rhizobacteria-induced ISR has been found to associate with Fe deficiency responses. Arabidopsis MYB72 not only activates the onset of ISR, but also increase plant survival under Fe deficiency (Zamioudis et al., 2014, 2015). To evaluate if BFKC01 stimulated ISR in Arabidopsis plants, assays of plant-bacteria interaction were performed. 12-day-old seedlings were inoculated with BFKC01 for different times, and the expression of some defense-related genes including PR1, PR2, and PDF1.2 was analyzed in the control and BFKC01-inoculated plants using RT-PCR (Fig. 7). Transcription of PR1 gradually increased following the time-delay of BFKC01 treatments, and the expression of other defense-related genes also exhibited the similarly changing tendency. Furthermore, the transcription levels of MYB72 were quantified in both the control and BFKC01-inoculated plants. The MYB72 transcripts significantly accumulated starting at 2 d of BFKC01 treatment and its transcription levels remained relatively high during the bacteria-plant interaction. 3.8. BFKC01 enhances biosynthesis and secretion of phenolic compounds

0 0h

6h

12 h

24 h

48 h

Fig. 2. Analyses of BFKC01-producing IAA in culture medium and endogenous IAA in Arabidopsis plants. (A) The concentration of IAA in bacterial cultures was quantified by HPLC. (B) Determination of endogenous IAA in the control and BFKC01-inoculated plants. 12-day-old Arabidopsis seedlings with or without inoculation of BFKC01 were grown on MS agar plates with 50 mM Fe(III)-EDTA for the indicated times, then these plants were used to measure the concentration of IAA. Data were shown as the means ± SE of at least three replicates, and the bars with asterisks indicated significant differences between control and BFKC01-incoulated plants using a Student's t-test at P < 0.05.

Fe deficiency causes a significant decrease in chlorophyll content, and reduces plant PSII activity. In this study, the Fv/Fm, an indicator for the efficiency of PSII photochemistry, was determined by measuring the chlorophyll fluorescence under growth light (120 mmol m2 s1). As shown in Fig. 5C, the ratios of Fv/Fm in the control plants were distinctly lower than those of the BFKC01inoculated plants. The altered tendency of actual PSII photochemical efficiency (FPSII) was similar to that observed in the Fv/Fm (Fig. 5D). The high chlorophyll fluorescence parameters in the BFKC01-inoculated plants were correlated with the improved photosynthesis and enhanced activities of PSII. 3.6. BFKC01 up-regulated the expression of FIT1, FRO2 and IRT1 in plants Arabidopsis FIT1 plays a central role in Fe homeostasis under Fe deficiency, which is essential for induction of IRT1 and FRO2 (Colangelo and Guerinot, 2004). To examine if BFKC01 regulated FIT1-mediated Fe deficiency responses, the transcription levels of FIT1 were analyzed in the control and BFKC01-inoculated plants using quantative PCR (qPCR). After 8 dpi, the transcription of FIT1 was up-regulated at least 6-fold in the BFKC01-inoculated plants grown on MS agar plates containing 5 mM, 20 mM or 50 mM Fe(III)EDTA, respectively (Fig. 6A). The expression of IRT1 and FRO2 was up-regulated more than 5-fold in the BFKC01-inoculated plants as

Overexpression of MYB72 promotes the production of fluorescent phenolic compounds via activation of the phenylpropanoid pathways (Zamioudis et al., 2014). These phenolic compounds can be secreted into the rhizosphere, facilitating plant uptake of Fe under low Fe-availability conditions (Schmid et al., 2014; Pii et al., 2015). In the present study, 12-day-old Arabidopsis seedlings were inoculated with BFKC01 and co-cultured for 2 d, 4 d and 8 d, respectively. Expression levels of genes involved in the phenylpropanoid pathways were analyzed in the control and BFKC01inoculated plants using qPCR. The transcription of these genes accumulated following BFKC01 treatments as compared to the control plants (Fig. 8). Also, the BFKC01-inoculated plants displayed stronger fluorescence in the vicinity of roots than control plants (Fig. 9A). Furthermore, the effect of BFKC01 on the production of root-secreted phenolic compounds was validated by a 96-well plate assay, in which the fluorescence emitted by root exudates was quantified. As shown in Fig. 9B, the BFKC01-inoculated plants produced more fluorescent phenolic compounds than control plants under alkaline conditions. Very low levels of fluorescent phenolic compounds were detected in the medium containing only BFKC01. 4. Discussion Fe is the fourth richest in the earth's crust, but Fe deficiency is an ubiquitous problem in world agriculture due to low Fe availability (Abadía et al., 2011). Many studies have shown that application of PGPRs improves plant uptake of nutrient elements such as nitrogen and Fe (Dey et al., 2004; Zhang et al., 2009; Pii et al., 2015), suggesting that beneficial rhziobacteria can be used as biofertilizers to improve plant nutrient status. P. polymyxa is one of well-known plant rhizosphere-colonizing bacteria that have been potentially used as biofertilizers or biocontrol agents (Anand et al., 2013; Park et al., 2014; Xu and Kim, 2014). In this study, the promoting effect of P. polymyxa strain BFKC01 on plant Fe assimilation is observed,

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Fig. 3. DR5:GUS signals and utrastructure analyses of root tissues in the control and BFKC01-inoculated Arabidopsis plants grown on MS agar plates with 50 mM Fe(III)-EDTA for 48 h (Control plants, A,C and E; Inoculated plants, B, D and F) DR5 in the tip of primary roots (A or B), vascular parenchyma in primary roots (C or D), the tip of lateral roots (E or F). In addition, strong DR5 signal occurred in differentiated tissues (arrow) of primary roots or the base of lateral roots from the BFKC01-inoculated plants (A or F). Utrastructure analyses of root cross-sections in vascular tissues of both the control (G) and BFKC01-inoculated plants (H).

which is mainly attributed to improvement of root system and activation of Fe-deficiency signaling pathways. Furthermore, BFKC01 obviously elevates the photosynthetic capacity of plants through increased chlorophyll content and photosynthetic efficiency. 4.1. BFKC01 improved root systems via activation of auxinmediated lateral root formation To sustain normal growth and development, most plants have developed robust root systems to extract water and mineral nutrients from soils. Several strains of rhizobacteria can affect root system architecture by interfering with plant hormone pathways

involving the regulation of root development (Moubayidin et al., 2009). Exogenous IAA can stimulate the formation of lateral roots and root hairs (Patten and Glick, 2002). In the present study, BFKC01-inoculated Arabidopsis plants had more lateral roots than control plants, suggesting that BFKC01 might produce and secrete IAA into plant rhizosphere. As expected, the concentration of IAA was detected in the culture supernatant of BFKC01. Additionally, a significant increase in endogenous IAA was also observed in the BFKC01-inoculated plants relative to the control plants. The DR5:GUS reporter system was used to visualize the localization of IAA and study auxin responses. BFKC01-treated plants showed a wide staining region in the vascular parenchyma, root tips and differentiated tissues of the primary roots, while only

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Fig. 4. Transmission electron micrographs of chloroplast ultrastructure in mesophyll cells of the control (AeC) and BFKC01-inoculated Arabidopsis plants (DeF). 12-day-old seedlings with or without inoculation of BFKC01 were grown on soils supplemented with different percent (w/w) of CaO for 8 days. Then, leaves of these plants was separated and used for TEM analyses. (A, D), 0% Cao; (B, E), 0.3% Cao; (C, F), 0.6% Cao. Bar ¼ 0.5 mm.

A *

3 Chlorophyll content ( mg g -1 FW)

B Control BFKC01

*

2.5 2

*

1.5 1

Pn (μmol m-2 s-1)

3.5

0.5 0 0.0%

0.3%

0.6%

20 18 16 14 12 10 8 6 4 2 0

C0 C0.3 C0.6

0

500

CaO (w/w)

D *

Control

*

0.6

BFKC01

1500

0.2

Control

*

BFKC01

*

0.6

*

0.4

1

0.8

ΦPSII

Fv/Fm

0.8

1000 PFD (μmol m-2 s-1)

C 1

T0 T0.3 T0.6

0.4

*

0.2

0 0.0%

0.3% CaO (w/w)

0.6%

0 0.0%

0.3%

0.6%

CaO (w/w) Fig. 5. Photosynthetic traits of both the control and BFKC01-inoculated Arabidopsis plants. 12-day-old seedlings with or without inoculation of BFKC01 were grown on soils supplemented with different percent (w/w) of CaO for 8 days. Then, these plants were used to measure total chlorophyll content (A), Pn (B), Fv/Fm (C) and FPSII (D). C0, C0.3 or C0.6, control plants grow in non-calcareous, 0.3% Ca0 or 0.6% CaO soils, respectively; T0, T0.3 or T0.6, BFKC01-inoculated plants grow in non-calcareous, 0.3% Ca0 or 0.6% CaO soils, respectively. Data were shown as the means ± SE of at least three replicates, and the bars with asterisks indicated significant differences between control and BFKC01-incoulated plants using a Student's t-test at P < 0.05.

weak staining was seen in the vascular parenchyma and root tip of control plants. A similar observation was made when Arabidopsis plants were treated with Phyllobacterium brassicacearum STM196 (Contesto et al., 2010). Auxin can activate initiation of pericyle

founder cells to produce lateral root primordia that continuously grow and emerge through the epidermal layers of the primary root (Guseman et al., 2015). A mass of cells was induced to divide in the root vascular tissues of the BFKC01-inoculated plants, whistle only

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Fig. 6. Effects of BFKC01 on the expression of FIT1, IRT1 and FRO2, and the ferric reductase activities in both the control and BFKC01-inoculated Arabidopsis plants. 12-day-old seedlings with or without inoculation of BFKC01 were grown on MS agar plates with different concentrations of Fe(III)-EDTA for 8 days, then total RNA were extracted from these plants to analyze the expression of FIT1 (A), IRT1 (B) and FRO2 (C) by qRT-PCR. Simultaneously, the reductase activities were quantified in both the control and BFKC01-inoculated plants (D). The ferric reductase activities (purple color) were observed greater in roots of the bacteria-inoculated plants than that in the control plants (E). Data were shown as the means ± SE of at least three replicates, and the bars with asterisks indicated significant differences between control and BFKC01-incoulated plants using a Student's t-test at P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

fewer cells divided in the controls. These findings strongly indicated that the production of IAA by BFKC01 could activate the auxin-signaling pathways to promote lateral root formation. 4.2. BFKC01 enhanced photosynthetic efficiency via increased chlorophyll content and promoted chloroplast development Low Fe-availability in alkaline or calcareous soils often induces Fe-deficiency chlorosis and interruption of chloroplast development (Abadía et al., 2011). In the present study, the Arabidopsis plants grown in alkaline conditions exhibited the severe chlorotic symptoms of yellowing leaves and low chlorophyll content. Similar phenomenon have been observed in other plant species such as maize and rice grown under Fe deficiency (Briat et al., 2007; Chen et al., 2015). Inoculation with BFKC01 increased the chlorophyll content in Arabidopsis plants grown under alkaline conditions. Similarity, inoculation with Azospirillum brasilense rapidly induced a significant increase in the chlorophyll content of

cucumber plants grown in calcareous soils (Pii et al., 2015). Our data revealed that normal chloroplast development was severely impaired in Arabidopsis leaves under low Fe-availability conditions. Few photosynthetic lamellae and grana were present in the chloroplasts of plants grown under Fe deficiency (Muneer et al., 2014). However, chloroplasts in leaves of the BFKC01-inoculated plants had more normal grana and grana lamellae than control plants. Fe deficiency often leads to disintegration of the lamellar network inside the chloroplast, and affects leaf photosynthetic capacity (Chen et al., 2015). In the present study, the photosynthetic rate (Pn) was remarkably elevated by BFKC01 under alkaline conditions, indicating that BFKC01 increased photosynthetic efficiency in Arabidopsis plants. Analyses of photosynthetic traits showed that Fv/Fm was also significantly enhanced in BFKC01-inoculated plants grown under alkaline conditions as compared to control plants. These results suggested that BFKC01 promoted the biogenesis of chloroplasts by increasing grana lamellae and hlorophyll levels, and

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Fig. 7. RT-PCR analyses of some marker genes involved in the ISR responses and biosynthetic gene of the phenylpropanoid pathway in both the control and BFKC01inoculated Arabidopsis plants. 12-day-old seedlings with or without inoculation of BFKC01 were grown on MS agar plates with different concentrations of Fe(III)-EDTA for the indicated times, then total RNA were extracted from these plants to analyze the expression of PR1, PR2, PDF1.2 and MYB72.

improved photosynthesis in the Arabidopsis plants. 4.3. BFKC01 enhanced Fe assimilation by activating FIT1-mediated deficiency responses Plants have evolved a set of complicated molecular regulatory mechanisms to adapt to Fe-deficiency stress. Several Fe homeostasis-regulated genes play essential roles in Fe uptake (Santi and Schmidt, 2009). In this study, inoculation with BFKC01 the chlorotic symptoms of Arabidopsis plants with increased Fe under alkaline conditions, implying that BFKC01 was sufficient to

trigger Fe deficiency responses, and accordingly improved the capacity of plants to absorb Fe. Our data revealed that the transcription level of AtFIT1, a master regulator for controlling Fe deficiency responses (Colangelo and Guerinot, 2004), was quickly induced in Arabidopsis plants after inoculation of BFKC01. Moreover, the expression of IRT1 and FRO2 substantially increased in the BFKC01-inoculated plants along with upregulation of FIT1. The observation was similar to the results reported by Zhang et al. (2009), Bacillus subtilis GB03 caused a significant increase in the expression of IRT1 and FRO2 via the FIT1-mediated pathways. In strategy I plants, Fe3þ can be reduced to Fe2þ in plant rhizosphere by the FRO2, the Fe2þ is then transported into root cells via the IRT1. Moreover, FRO2-mediated ferric reduction has been shown to be a key rate-limiting step in Fe uptake under Fe deficiency (Zhang et al., 2009). Therefore, abundant accumulation of IRT1 and FRO2 transcripts induced by BFKC01 was conducive to plant Fe uptake under Fe deficiency. 4.4. BFKC01 stimulated fluorescent phenolic compounds to promote Fe uptake Induced systemic resistance (ISR) is an intricate phenomenon in which immune elicitors from several soil microbes differentially activate a series of signal transduction pathways participating in disease resistance (Lee et al., 2012; Zamioudis et al., 2015). In this study, the expression of some defense-related genes including PR1, PR2 and PDF1.2 were significantly up-regulated in the BFKC01inoculated plants, indicating that this bacteria could stimulate plant ISR. Similar results have been observed in previous studies, several strains of beneficial rhizobacteria such as P. polymyxa E681 and Bacillus amyloliquefaciens IN937a trigger ISR in plants (Lee et al., 2012; Mei et al., 2014). Rhziobacteria-mediated ISR in plants has been shown to correlate with Fe deficiency responses. Zamioudis et al. (2015) have shown that ISR-inducing bacteria WCS417 can trigger broad spectrum resistant gene responses in Arabidopsis

Fig. 8. qRT-PCR analyses of the expression levels of the phenylpropanoid biosynthetic genes in both the control and BFKC01-inoculated Arabidopsis plants. 12-day-old seedlings with or without inoculation of BFKC01 were grown on MS agar plates with 50 mM Fe(III)-EDTA for the indicated times, then total RNA were extracted from these plants for analyzing the expression of the phenylpropanoid biosynthetic genes. (A) Schematic of the phenylpropanoid biosynthetic pathways, some key enzymes were labeled in red. (B) The expression levels of the corresponding genes encoding these enzymes were analyzed by qRT-PCR. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate: CoA ligase; HCT, hydroxycinnamoyl-coenzyme A shikimate: quinate hydroxycinnamoyl-transferase; C30H, p-coumaroyl shikimate 30 hydroxylase; CCoAOMT, caffeoyl CoA 3-Omethyltransferase; F60 H1, feruloyl-CoA 60-hydroxylase1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 9. Analyses of fluorescent phenolic compounds secreted by the control and BFKC01-inoculated Arabidopsis plants. 12-day-old seedlings with or without inoculation of BFKC01 were grown on MS agar plates or liquid MS medium (pH ¼ 7.2) with different concentrations of Fe(III)-EDTA for 8 days. (A) Visualization of fluorescent phenolic compounds that are secreted by roots of the control and BFKC01-inoculated plants grown under conditions of 5 mM, 20 mM or 50 mM Fe(III)-EDTA. Photographs were taken under UV lamps with a wave length of 365 nm excitation. (B) Relative quantification of fluorescent phenolic compounds in the liquid MS medium of BFKC01, control plants or BFKC01-inoculated plants grown in a 9-well plate under the indicated conditions. After removing BFKC01 or plants from the growth medium, fluorescence was quantified in a 96-well microplate reader. Data were shown as the means ± SE of at least three replicates, and the bars with aterisks indicated significant differences among different groups (BFKC01, Plants, and Plants þ BFKC01) using a Student's t-test at P < 0.05.

roots, and the majority of these genes are also regulated by the root-specific MYB72. These findings indicates that this gene functions as a key linker protein between ISR and Fe uptake in plants. Overexpression of MYB72 in Arabidopsis plants up-regulated the expression of genes encoding biosynthetic enzymes of the phenylpropanoid pathway and promoted the biosynthesis of phenolic compounds (Zamioudis et al., 2014). Under Fe deficiency, phenolic compounds can be secreted by plant roots to mobilize Fe from the rhizosphere. Interestingly, cucumber plants inoculated with Azospirillum brasilense exhibited increased soil release of phenolic compounds under Fe deficiency (Pii et al., 2015b), indicating that rhizobacteria can enhance Fe assimilation by promoting root-secreted phenolic compounds. In this study, the transcription of MYB72 was significantly increased in BFKC01inoculated plants relative to control plants. Additionally, the expression of genes involved in the phenylpropanoid pathways was significant higher in BFKC01-inoculated plants than control

plants. More fluorescent phenolic compounds were found in the rhizosphere of BFKC01-inoculated plants than in the control plants. These results indicate that BFKC01 can activate MYB72mediated ISR, and promote the production of Fe-mobilizing phenolic compounds. Based on data from this study, the following model of microbial enhancement of plant Fe assimilation by BFKC01 is proposed. As illustrated in Fig. 10, microbial production of IAA by BFKC01 activates auxin-mediated signaling pathways and promotes lateral root formation, so plants efficiently absorb Fe from the rhizosphere. BFKC01 regulates plant Fe uptake by integrating the mechanisms of both enhancement of Fe deficiency responses and increased secretion of iron-mobilizing phenolic compounds. This research provides evidence that BFKC01 can increase the bioavailability of Fe in alkaline or calcareous soils, and demonstrates its potential roles in promoting plant Fe assimilation.

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BFKC01 Activation of MYB72 BFKC01 Auxin FeII FeIII

Activation of auxin-mediated signaling pathways Promoted formation of lateral roots

FeIII eIIII

BFKC01

FeIII Activation A c of FIT1 Fe(III) FRO2

IRT1 Fe(II) Epidermic cells

Fig. 10. A proposed model of BFKC01-improved plant Fe assimilation. BFKC01 can produce and secrete IAA, which improves plant root systems. Additionally, certain metabolites secreted by BFKC01 stimulate FIT1-mediated Fe acquisition via up-regulation of FRO2 and IRT1, and activate MYB72-mediated phenolic compounds that effectively mobilize Fe from plant rhizosphere.

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