Antioxidant enzymes in plant can remove ROS and alleviate oxidative damage (Krantev et al., 2008; Mishra et al., 2011). It has been known that the higher activities of CAT and SOD could improve plant tolerance to salinity and K+-deficiency conditions (Wang et al., 2010; Zhou et al., 2014). It was found that SOD activity was significantly higher in the leaves of salttolerant wild tomato (*Lycopersicon pennellii*) than that of saltsensitive cultivated tomato (*Lycopersicon esculentum*) after 12 and 84 d of salt treatment (140 mM NaCl) (Koca et al., 2006). Similarly, salt-tolerant *Plantago maritima* showed a better protection mechanism against oxidative damage caused by salt stress by its higher induced activities of CAT, SOD, glutathione reductase (GR) and peroxidase (POX) than the salt-sensitive *P. media* (Sekmen et al., 2007). Co-expression of the *Suaeda salsa* CAT and glutathione S-transferase (GST) genes enhanced the active oxygen-scavenging system that led to improved salt tolernace in transgenic rice, resulting from not only increased CAT and GST activities but also the combined increase in SOD activity (Zhao and Zhang, 2006). Jing et al. (2014) reported that overexpression of mangrove (*Kandelia candel*) copper/zinc superoxide dismutase gene (*KcCSD*) enhanced salinity tolerance in tobacco: *KcCSD*transgenic lines were more Na+ tolerant than wild-type (WT) tobacco in terms of lipid peroxidation, root growth, and survival rate; Na+ injury to chloroplast was less pronounced in transgenic tobacco plants due to enhanced SOD activity by an increment in SOD isoenzymes under 100 mM NaCl stress from 24 h to 7 d; catalase activity rose in *KcCSD* overexpressing tobacco plants and transgenic plants better scavenged NaCl-elicited ROS compared to WT ones. In present study, the activities of CAT and SOD in leaves of potato plantlets significantly increased with the increase of NaCl concentration (0∼100 mM) in medium. When exposed to 200 mM NaCl, especially after 6 weeks, leaf cells were severely damaged, even disorganized (**Figure 1**), leading to the damage of cellular structure or alterations of metabolism, and reducing the synthesis of CAT and SOD.

Soil salinity is known to increase the level of ROS in plant leaves and MDA is a major product of membrane lipid peroxidation (Mittova et al., 2004; Koca et al., 2006; Yazici et al., 2007). Therefore, leaf MDA content could represent the degree of cell membrane damage and is usually used to evaluate plant salt tolerance (Luna et al., 2000; Miao et al., 2010; Han et al., 2014). In our study, leaf MDA content increased significantly with the increase of external salt concentration after 2-week treatment and even increased more rapidly after 6-week treatment. However, the activities of SOD and CAT may not enough to eliminate ROS, resulted in large production of MDA under higher salt stress (200 mM).

### **SALINITY REDUCED LEAF STOMATAL AREA AND CHLOROPHYLL CONTENT**

Chlorophyll is essential for photosynthesis, and the increase of chlorophyll content can reflect the increase of photosynthetic activity (Yamori et al., 2006). Ben et al. (2010) and Su et al. (2011) suggested that the accumulation of chlorophyll content could enhance plant salt tolerance. In the present study, leaf chlorophyll content gradually decreased with the increase of NaCl treatment and duration, which could result from the inhibition of chlorophyll synthesis caused by chloroplast damage.

Gas exchange through stoma play important role in carbon assimilation (Wilkinson and Davies, 2002). Salt stress decreases leaf stomatal area by reducing leaf water content and leaf turgor induced by ABA signal (Wilkinson and Davies, 2002). Therefore, stomatal conductance was correlated to salinity stress (Liu et al., 2014b). In our study, salt stress seriously induced stomatal closure. Reduced CO2 diffusion caused by stomatal closure lead to suppression of photosynthesis, affecting plant growth (**Figure S1**).

In conclusion, the adaptation of plants to salt stress is a complex process at cellular, biochemical and physiological levels. In the present study, several parameters were analyzed to demonstrate ultrastructural and physiological responding mechanisms of potato (*Solanum tuberosum* L.) plantlets to gradient saline stress (**Figure 8**). We found that with the increase of external NaCl concentration and the duration of treatments, the number of chloroplasts and cell intercellular space markedly decreased, cell wall thickened and even ruptured, and mesophyll cells and chloroplasts were gradually damaged to a complete disorganization. Above ultrastructural changes may be induced by the increased concentrations of Na+ that was transported into cytosol probably through non-selective cation channels (NSCCs), high-affinity K+ transporters (HKTs, probably HKT1;2; HKT1;4; HKT1;5 and HKT2;1) and permeated directly across plasma membrane, and Cl− that was probably transported by cation-Cl− cotransporter (CCC) (Apse and Blumwald, 2007; Plett and Moller, 2010; Zhang et al., 2010; Zhang and Shi, 2013; Almeida et al., 2014a,b; Maathuis, 2014; Maathuis et al., 2014). More and more K+ was probably transported out of the cell by K+ outward-rectifying channels (KORs) activated by membrane depolarization (DPZ) (Chen et al., 2007; Sun et al., 2009; Lu et al., 2013; Demidchik, 2014; Demidchik et al., 2014; Lai et al., 2014). Leaf MDA content increased significantly

due to all membrane lipid peroxidation induced by increasing and continuous salt stress, which also induced stomata closure and chlorophyll content decline. Potato plantlets showed adaptation ability to moderate salt stress through Na+ efflux or extrusion by plasma membrane Na+/H+ antiporter (salt overly sensitive, SOS1) motivated by plasma membrane ATPase (PM-ATPase), vacuolar Na+ compartmentation by tonoplast Na+/H+ antiporter (NHX1) driven by vacuolar ATPase (V-ATPase) and H+-pyrophosphatase (VP1), accumulating osmoprotectants such as proline, and improving the activities of antioxidant enzymes (CAT and SOD). This work provided both anatomical and physiological data for characterization of damages induced by salinity and the method could be used for selecting salt-tolerant potato cultivars.

### **ACKNOWLEDGMENTS**

This research was supported by Program for Changjiang Scholars and Innovative Research Team in University (IRT13019), International Science & Technology Cooperation Program of China (2014DFG31570), Gansu S&T Foundation (1308RJZA131 and 1308RJIA005), Lanzhou S&T Research Project (2013-4-156 and GSCS-2012-04) and NSFC (31222053).

### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: http://www*.*frontiersin*.*org/journal/10*.*3389/fpls*.*2014*.* 00787/abstract

**Figure S1 | Growth of potato plantlets in MS agar plates.** Plantlets grown on MS were transferred to new solid agar MS supplemented with various concentrations of NaCl (0, 25, 50, 100, and 200 mM) for 2 and 6 weeks, respectively.

### **REFERENCES**

gas exchange and leaf ultrastructure of *Arbutus unedo* plants. *Plant Sci*. 172, 473–480. doi: 10.1016/j.plantsci.2006.10.006

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 21 August 2014; accepted: 18 December 2014; published online: 13 January 2015.*

*Citation: Gao H-J, Yang H-Y, Bai J-P, Liang X-Y, Lou Y, Zhang J-L, Wang D, Zhang J-L, Niu S-Q and Chen Y-L (2015) Ultrastructural and physiological responses of potato (Solanum tuberosum L.) plantlets to gradient saline stress. Front. Plant Sci. 5:787. doi: 10.3389/fpls.2014.00787*

*This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science.*

*Copyright © 2015 Gao, Yang, Bai, Liang, Lou, Zhang, Wang, Zhang, Niu and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Light as stress factor to plant roots – case of root halotropism

### *Ken Yokawa1,2 , Rossella Fasano3 , Tomoko Kagenishi 1 and František Baluška1\**

1 Department of Plant Cell Biology, Institute of Cellular and Molecular Botany, University of Bonn, Bonn, Germany

2 Department of Biological Sciences, Tokyo Metropolitan University, Tokyo, Japan

3 Department of Pharmacy, University of Salerno, Fisciano, Italy

#### *Edited by:*

Vadim Volkov, London Metropolitan University, UK

### *Reviewed by:*

Francois Bouteau, Université Paris Diderot, France Zheng-Hui He, San Francisco State University, USA

#### *\*Correspondence:*

František Baluška, Department of Plant Cell Biology, Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Germany e-mail<EMAIL_ADDRESS>
Despite growing underground, largely in darkness, roots emerge to be very sensitive to light. Recently, several important papers have been published which reveal that plant roots not only express all known light receptors but also that their growth, physiology and adaptive stress responses are light-sensitive. In Arabidopsis, illumination of roots speedsup root growth via reactive oxygen species-mediated and F-actin dependent process. On the other hand, keeping Arabidopsis roots in darkness alters F-actin distribution, polar localization of PIN proteins as well as polar transport of auxin. Several signaling components activated by phytohormones are overlapping with light-related signaling cascade. We demonstrated that the sensitivity of roots to salinity is altered in the light-grownArabidopsis roots. Particularly, light-exposed roots are less effective in their salt-avoidance behavior known as root halotropism. Here we discuss these new aspects of light-mediated root behavior from cellular, physiological and evolutionary perspectives.

**Keywords: root, light response, plant hormones, reactive oxygen species, root tropism**

### **LIGHT AS IMPORTANT ENVIRONMENTAL FACTOR FOR ROOTS**

In nature, sessile plants have to respond to diurnal change in the light environment. One of main roles of light in plant's life is to provide energy for photosynthesis and for the regulation of plant development at different stages such as seed germination, vegetative growth, tropisms and flowering. It is known that plant photoreceptors and related light-sensitive signaling molecules participate in the regulation of physiological conditions and morphological plasticity in response to the light environment. Darwin (1879) has discovered negative phototropism of plant roots. One year later, Francis Darwin and his father, Charles Darwin, published the book,"The Power of Movements in Plants." They described both root and shoot tropisms. In addition, they also proposed that some form of long-distance signaling connect the sensory organ apices with the actively growing basal parts (Darwin, 1879, 1880). Since then, dedicated research work in plant physiology has discovered the long-distance signaling molecule, auxin, resulting in insights into plant photoreception. This directional growth response to incoming light is called phototropism. Positive phototropism, observed in shoots, is growth toward a light source; whereas negative phototropism, seen in roots, is bending away from the light source. We demonstrated that short (10 s), but strong (the photon flux was 82 μmol m−2 s −1), blue light illumination of *Arabidopsis* roots induces the immediate generation of reactive oxygen species (ROS) in root apex region, resulting in rapid increase of the root growth rate (Yokawa et al., 2011, 2013). This active response of light-stimulated root growth is termed escape tropism (Xu et al., 2013; Yokawa et al., 2013; for maize roots see Burbach et al., 2012). This tropism would allow *Arabidopsis* roots to escape from unfavorable light conditions if growing outside of our laboratories in the nature.

### **PHOTORECEPTORS IN ROOTS**

It has been shown that *Arabidopsis* plant expresses 14 photoreceptors, most of which are also present in roots (Briggs and Lin, 2012; Jeong and Choi, 2013; Briggs, 2014). Roots grow in the dark soil to anchor the plant and to absorb mineral nutrients and water. It has been reported that light can penetrate less than several millimeters due to the rather high absorbance of soil (Woolley and Stoller, 1978). Nevertheless, small cracks or mechanical impacts can often happen which allows light to penetrate deeper. For instance, roots may be exposed to light due to sudden temperature changes, earthquake, heavy rain, wind, and so on. In addition, it is very important for emerging radicle to increase the root growth rate shortly after seed germination on the ground. It was necessary to evolve the ability of roots to respond to environmental light when the first flowering plants with modern root system emerged in land plant evolution. In the next section, intriguing interplays between phytohormones and light-related signaling pathways will be discussed.

### **FROM ACTIN CYTOSKELETON, VIA PIN2 RECYCLING, TO SALT AVOIDANCE TROPISMS OF ROOTS**

At the cellular level, it was reported that PIN2 proteins (PIN-FORMED 2; auxin efflux carrier) in root apices respond to the light environment (Laxmi et al., 2008). Wan et al. (2012) demonstrated that the basipetal (shootward) PIN2-based polar auxin transport is subject to blue light control, which regulates the negative phototropism of *Arabidopsis* roots (Wan et al., 2012). Moreover, Dyachok et al. (2011)reported that light-activated COP1, E3 ubiquitin ligase, promotes actin polymerization and F-actin bundling, through regulation of the downstream ARP2/3-SCAR pathway in root cells. It results in increased root growth under the illuminated conditions (Dyachok et al., 2011). It was also reported

that light controls bundling of F-actin in maize coleoptiles (Waller and Nick, 1997), changing sensitivity of cells to auxin, which is feeding back to control F-actin as well as cell growth (Nick et al., 2009). The interplays between F-actin and polar auxin transport mediated by endocytic vesicle recycling, especially in the transition zone of root apex, control root tropisms (Baluška et al., 1996, 2004, 2005, 2010; Baluška and Mancuso, 2013). Interestingly, precursor of endogenous auxin, indole-3-acetaldehyde (IAAld), is produced non-enzymatically *in vitro* by illumination of tryptophan in the presence of flavin which is abundant in living plant cells (Koshiba et al., 1993). Recently, we have proposed close links between the redox status and auxin (IAA) biosynthesis in plants (Yokawa et al., 2014). Taken together, it is obvious that roots are extraordinarily sensitive to light exposure due to their inherent evolutionary optimization for the underground life. Therefore, it is not surprising that illuminated roots of young *Arabidopsis* seedlings enhance their growth with the concomitant phototropism.

A few yeas ago, salt-stressed roots of *Arabidopsis* have been shown to alter root growth direction in order to avoid high salt areas via so-called salt avoidance tropism (Li and Zhang, 2008; Sun et al., 2008). This active root tropism requires ion gradient sensing pathway which would then control the PIN2 abundance, recycling and degradation (Li and Zhang, 2008; Sun et al., 2008). This unique *Arabidopsis* root behavior was linked to phospholipase D Zeta2 (PLDζ2) activity which stimulates clathrin-mediated endocytosis of PIN2, and this tropism was also termed root halotropism (Galvan-Ampudia et al., 2013; Rosquete and Kleine-Vehn, 2013; Pierik and Testerink, 2014). Interestingly, similarly, as in halotropism, PLDζ2 is involved in root hydrotropism through the PIN2-mediated suppression of root gravitropism (Taniguchi et al., 2010). Moreover, PLDζ2 is crucial for brefeldin A-sensitive endocytic recycling driving PIN2 recycling (Li and Xue, 2007) and polar auxin transport in the transition zone (Mancuso et al., 2007). Because PIN2 is crucial in this respect, as well as in adaptive responses of roots to light (Laxmi et al.,2008; Sassi et al.,2012;Wan et al., 2012), it is very important to test whether light condition affect the response of *Arabidopsis* roots to the salt stress.

### **PLANT HORMONES ARE INTEGRATED WITH LIGHT SIGNALING PATHWAYS**

Karrikin molecule was isolated from the smoke of combusted plant materials and found to potently stimulate the germination of plant seeds (Flematti et al., 2004). This compound was identified as 3 methyl-2H-furo[2,3-c]pyran-2-one, karrikin-1 or karrikinolide-1 (KAR1) shown in **Figure 1A**-a, and another analogs of karrikins (KAR2-KAR6) commonly possess butenolide structure. The *Arabidopsis* mutant *kai2* lacking *KAI2* genes insensitive to KAR1 triggered promotion of seed germination. KAI2 is thought to be a putative receptor of karrikins (Nelson et al., 2010; Waters et al., 2012). KAI2 is a member of α/β-hydrolasefamily and is also known as HYPOSENSITIVE TO LIGHT (HTL). Role of KAI2/HTL in light responses (**Figure 1B**) of roots will be described in the next section.

*Arabidopsis thaliana* AtD14 (ortholog of DWARF14) is a paralog of KAI2/HTL, which belongs to α/β-hydrolase family and plays a role in strigolactone perception (Waters et al., 2012).

Strigolactones are synthesized from carotenoids and released into root exudates to promote germination of parasitic weeds (Yoneyama et al., 2007), as well as to stimulate hyphal branching in arbuscular mycorrhizal fungi (Akiyama et al., 2005). Strigolactones act also as a new class of plant hormone that inhibit the growth of axillary buds (Umehara et al., 2008) and alter root architecture (Koltai, 2011). Strigolactones are categorized as a sesquiterpenes lactone and have two moieties of butenolide (shown in **Figure 1A**-b as synthesized analog, GR24). As described above, karrikins have butenolide moieties as well. Therefore, because of the structural similarity of two molecules, karrikin receptor KAI2/HTL can respond to strigolactones. However, in contrast, the proposed strigolactone receptor AtD14 is insensitive to karrikins; indicating that the activity of KAI2/HTL as α/β-hydrolase is more flexible to detect butenolide structure than AtD14, probably due to evolutionary issues (Waters et al., 2012).

It was reported that F-box protein, MORE AXILLARY BRANCHES2 (MAX2), is located in the downstream of both KAI2/HTL and AtD14 and thus necessary for plants to respond to karrikin and strigolactone (Nelson et al., 2010; Waters et al., 2012). MAX2 forms SCF E3 ubiquitin ligase complex (Skp1, Cullin1, F-box), which modulates further downstream transcriptions (Stirnberg et al., 2007; Shen et al., 2012). However, molecular functions of KAI2/HTL or AtD14-mediated SCFMAX2 regulation of plant morphogenesis are not known yet. Interestingly in this

respect, *max2* mutants are hyper-sensitive to drought and osmotic stress, including high NaCl, mannitol, and glucose (Bu et al.,2014). Furthermore, strigolactones exert positive roles in plant adaptation to drought and salt stress (Ha et al., 2014). We will discuss these newly emerging aspects in the final section.

### **LIGHT SIGNAL TRANSDUCTION VIA COP1 AND HY5 IS INTEGRATED WITH HORMONAL SIGNALING**

In principle, plants recognize light as specific wavelengths by specific photoreceptors. Activated (excited) photoreceptors convert the light information into physiological signaling in diverse manners. CONSTITUTIVE PHOTOMORPHOGENIC1(COP1), E3 ubiquitin ligase, participates in the light-perceiving signaling cascade via affecting ubiquitination of target proteins. Once the activity of COP1 is inhibited, the downstream bZIP transcription factor, ELONGATED HYPOCOTYL5 (HY5) is freed from ubiquitination by COP1 and starts specific gene transcriptions related to light responses (**Figure 1B** left). COP1 and HY5 control root growth, lateral root formation, root hair tip growth, as well as root touch-responses and gravitropism (Oyama et al., 1997; Ang et al., 1998; Cluis et al., 2004; Datta et al., 2006, 2007; Sibout et al., 2006). Importantly, SALT TOLERANCE HOMOLOG2 (STH2) is interacting partner of COP1 and HY5 which controls roots and their anthocyanin levels (Datta et al., 2007).

Interestingly, exposure of plants to strigolactone inhibits the COP1 activity, suggesting that strigolactones can mimick light perception in plants (Tsuchiya et al., 2010). They also demonstrated that *max2* mutant of rice produces excess of strigolactones, resulting in the inhibition of COP1 and expression of light-responsive genes (Tsuchiya et al., 2010). In addition, it was reported that *max2* mutant is hypersensitive to red, far-red and blue light (Shen et al., 2007). Auxin interaction with the signaling pathway of strigolactones via MAX2 was already reported (Hayward et al., 2009). Finally, the key transcription factor of light signaling HY5 requires strigolactones in order to stimulate *Arabidopsis* seed germination during thermoinhibiton (Toh et al.,2012). Light also induces auxin biosynthesis via photoexcitation of flavins (Koshiba et al., 1993; Yokawa et al., 2014).

Karrikin receptor KAI2 is also known as HTL. KAI2/HTL expression was strongly increased by blue, red and far-red light and HY5 binds to the promoter region of HTL, indicating the expression of KAI2/HTL is regulated in response to light environment (Sun and Ni, 2011). Furthermore, KAI2/HTL is located downstream of HY5 (**Figure 1B**) and induced by light treatment (Sun and Ni, 2011). Meanwhile, it was reported that the treatment of seeds with KAR1 (chemical structure is shown in **Figure 1A**-a) improved light responses in germination and early development of seedlings (Nelson et al., 2010). It implies that karrikins can affect the array of gene expression regarding light responses.

Plant roots exposed to salinity stress increase abscisic acid (ABA) and ABA-related gene expressions (Raghavendra et al., 2010). ABA and strigolactones are synthesized from carotenoids and the levels of these two plant hormones are affecting each other (López-Ráez et al., 2010). Salt stress increases strigolactone levels in roots which then promote arbuscular mycorrhizal symbiosis, resulting in changing ABA contents and alleviating stress response

(Aroca et al., 2013). Strigolactones, ABA and cytokinin signaling pathways are integrated to allow plants to cope with high salinity environment very effectively (Ha et al., 2014). Many players of the phytohormone signaling pathways are overlapping and integrating with light-response pathways.

Taken together, these findings suggest that hormonal and light signaling pathway utilizes same junction for plant morphogenesis as well as for adaptation to abiotic stresses. The signaling pathways proposed (**Figure 1B**) are highly integrated, interacting with each other and, obviously, they represent just a 'tip of the iceberg.' In the case of roots, we should consider light solely as information (not as source of energy) into our careful consideration for our understanding of plant photomorphogenesis. In the next session, we are discussing how the root sensitivity to salinity is altered via exposure of roots to light.

### **UV LIGHT AND UVR8 IN DROUGHT AND SALINITY RESPONSES OF ROOTS**

It has been reported that the reduction of plant growth under water deficit is driven by the UV-B photoreceptor UV RESIS-TANCE LOCUS 8 (UVR8; Kliebenstein et al., 2002; Brown et al., 2005; Favory et al., 2009; Fasano et al., 2014). Fasano et al. (2014) previously shown that the *UVR8* gene complements the osmosensitive yeast mutant *mpk1 ppz1*. The expression of *UVR8* was found upregulated in *Arabidopsis* plants grown under salt or osmotic stress conditions. Furthermore, the ectopic expression of *UVR8* causes pleiotropic effects on plant growth, such as a general reduction of plant organ size, leaves with smaller cells, reduced root growth, and the accumulation of flavonoids. This suggests that the UV-B morphogenic responses are enhanced in the *UVR8* overexpressing plants grown under low levels of UV-B light. The growth defects of the *UVR8*-overexpressing plants are even more severe under osmotic and salt stress. In contrast, the inactivation of *UVR8* expression does not affect shoot or root growth under standard or mild drought stress conditions. Thus, the hypersensitive response to osmotic stress of the *35SUVR8* plants is strictly UVR8-dependent (Fasano et al., 2014).

There are extensive evidences that osmotic stress as well as UV-B light impacts are more prominent on shoots than on roots. Under mild osmotic stress, roots continue to grow (Bartels and Sunkar, 2005; Pardo, 2010; Werner et al., 2010). Moreover, enhanced root growth was observed in transgenic plants with a higher drought or salt-stress tolerance (Bartels and Sunkar, 2005). Whereas the impact of osmotic stress on root development is well known (recently reviewed in Liu et al., 2014), effects of UV-B on root growth are poorly understood. In general UV-B reduces primary root growth of *Arabidopsis* seedling (Kim et al., 1998; Tong et al., 2008). In the adult plant, increased allocation of biomass to roots has been reported to occur under UV-B stress (Bussell et al., 2012).

Development of a larger root system is considered as a droughtavoidance strategy that plants adopt to improve the uptake of water and nutrients when their availability in the soil is limited (Fukai and Cooper, 1995; Liao et al., 2001; Sharp et al., 2004; Pierik and Testerink, 2014). The UV-B photoreceptor UVR8 is expressed in roots of wild type plants (Rizzini et al., 2011; reviewed inYokawa and Baluška, 2014). As shown in **Figure 2**, UVR8::GFP is

(3.9 μmol m−2 s−1).

expressing in*Arabidopsis* roots and is transported into nuclei upon irradiation of roots with UV-B. Over-expression of UVR8 greatly reduces root growth under light exposure and aggravates their weaker performance under the osmotic stress (Fasano et al., 2014). In comparison to the control plants, the primary root and lateral root densities were 13 and 60%, respectively. This indicates that auxin-dependent lateral root growth was most hampered. Indeed the root-phenotypes of the *35S-UVR8* plants are reminiscent that one of auxin mutant (Zhao, 2010). The analysis of lateral roots showed that the number of lateral root primordia and emerged lateral roots of the *35S-UVR8* plants was 12 and 68%, respectively, reduced in comparison to the control plants (Fasano et al., 2014). Flavonoids accumulation was found to be 2.2-fold increased in the root of the *UVR8*-over-expressing plants, whereas the content of IAA-conjugates showed a tendency to decrease (Fasano et al., 2014). Thus, the defects in cell expansion of the *35S-UVR8* roots could be associated to the increased levels of flavonoids which, in turn, alter polar auxin transport and/or auxin homeostasis (e.g., Santelia et al., 2008).

Flavonoids are synthesized in roots, regulating root branching, gravitropism, and stress adaptation (Brown et al., 2001; Buer and Muday, 2004; Agati et al., 2011; Emiliani et al., 2013). Flavonoid concentrations are significantly higher in combined stress-treated plants than in those treated with UV-B alone (Hughes et al., 2010; Comont et al., 2012). Together, all these reports suggest that *UVR8*, via the control of flavonoid accumulation, might be a common intermediate in light and hormone signaling pathways to regulate root growth and development under abiotic stress challenges. Besides UV-B, drought and salinity have also been associated with anthocyanin accumulations in various tissues including roots (Agati et al., 2011; Emiliani et al., 2013; Meng, 2014). Importantly in this respect, the light-induced stimulation of *Arabidopsis*

root growth is mediated via the COP1-mediated accumulation of anthocyanins (Meng, 2014).

### **IMPACT OF LIGHT ON SALINITY AVOIDANCE VIA ROOT HALOTROPISM**

Although root apices growing in soil are known to be at a front line that can be exposed to salt stress, an impact of light on modulation of the sensitivity of roots to salinity has not been considered to date. Importantly, most experiments using the laboratory grown *Arabidopsis* seedlings are using roots exposed to light, although root apices outside in the nature are mostly in darkness.

As shown in the **Figure 3**, we have demonstrated that light exposure affects the root response to the salinity stress. Columbia WT seedlings were grown on agar plates containing NaCl only at their bottom parts. With this system, root tropism responses to salinity, halotropism, can be observed. Our methods followed the protocol described in (Galvan-Ampudia et al., 2013; Rosquete and Kleine-Vehn, 2013). After replacing bottom part of agar with the NaCl-agar, plastic dishes were covered with aluminum foil to allow dark treatment. Light/dark period was 16 h/8 h and light intensity was about the 120 μmol m−2 s −1, comparable to conditions in Galvan-Ampudia et al. (2013). Both the darkand light-grown roots grew straight to the boundary of two agar media in control experiments with 0 mM NaCl (**Figure 3**). On the other hand, roots growing in darkness have higher sensitivity to NaCl than that roots exposed to light (**Table 1**). Dark-grown roots showed halotropism by avoiding salt-enriched agar areas at all concentrations of NaCl (**Figure 3**; **Table 1**). At the 100 mM concentration of NaCl, light-grown roots kept growing into the salty part of agar across the borderline between two agar parts even after several days. This suggests that light either prevents roots to accomplish the halotropism or that light lowers the sensitivity of *Arabidopsis* roots to perceive a gradient of salinity. The fact that light grown roots performed halotropism at higher concentrations of NaCl (**Table 1**) suggests that light interferes with the elusive root's ability to sense the Na+ gradients (Maathuis, 2014). As root apices of roots growing outside in the nature are typically located deep in the soil in darkness, it can be expected that their halotropism is more efficient than for roots of laboratory grown seedlings.

### **LIGHT-INDUCED ROOT GROWTH IS EXPENSIVE AND ALTERS PHYSIOLOGY AND MORPHOLOGY OF WHOLE SEEDLINGS**

It is important to be aware that light-induced stimulation of root growth is changing the physiology of whole seedlings/plants. **Figure 4** summarizes light-induced root growth mediated via COP1 interactions with the actin cytoskeleton (Dyachok et al., 2011) and anthocyanin biosynthesis (Meng, 2014). Both SCAR and PAP mediated pathways require sucrose as energy source (Kurata and Yamamoto, 1997; Kircher and Schopfer, 2012; Maier et al., 2013; Meng, 2014). Roots lacking ANGUSTIFOLIA3 (AN3) transcription coactivator have reduced anthocyanin levels and longer roots in light than WT roots (Meng, 2014). In contrast, the *an3* mutant roots are shorter that WT roots when grown in darkness. Interestingly, AN3 binds to COP1 promoter to inhibit the light-induced root elongation (Meng, 2014). Besides anthocyanins, light-exposed roots show also rather dramatic increase of

**FIGURE 3 | Light environment affects sensitivity of** *Arabidopsis* **roots to salinity.** Seedlings of Col-0 were firstly grown on 1/2 MS media for 5 days. Bottom part of agar was removed diagonally and new 1/2 MS media containing NaCl was added. Petri dishes were placed either in light or dark condition for 3 days. Roots growing on agar plates in dark/light conditions

with 0, 100, 200, and 300 mM NaCl media in the bottom half of Petri dishes. The method used for imposing the root halotropism is following exactly the protocol as published by Galvan-Ampudia et al. (2013). White arrows indicate the gravity vector. All seedlings were grown in vertical position. Bar indicates 1 cm.

**Table 1 | Halotropic bendings of** *Arabidopsis* **roots grown in different light conditions.**

Roots have been considered halotropic when scored root apex angles are greater than 45◦ with respect of the gravity vector (= same angle with the boundary of agar).

phenylpropanoid metabolism, inducing not only flavonoids but also monolignol glucosides (Hemm et al., 2004). Possible impacts of light on the Casparian bands formation in endodermis can be expected and easily tested.

Light-induced actin polymerization, auxin polar transport, root growth and anthocyanin biosynthesis are energetically demanding processes. The increased demand of sucrose for the root photomorphogenesis (Costigan et al., 2011; Warnasooriya and Montgomery, 2011; Yokawa et al., 2013) is going at the expense of other potential sinks of the plant body (Kurata and Yamamoto, 1997; Yazdanbakhsh et al., 2011; Kircher and Schopfer, 2012). Moreover, salt stress perceived locally at the root apex is rapidly spread throughout the plant body via systemic signaling (Choi et al., 2014; Gilroy et al., 2014). Therefore, it is important to be aware that illumination of roots has rather dramatic consequences for the whole seedling's physiology and morphology.

### **CONCLUSIONS AND PERSPECTIVES**

Our results using salt stress reveal that light exposed roots show different behavior and responses under salt stress. One possible scenario, based on reports that illuminated roots enhance

**Table 2 |** *Arabidopsis* **root lengths in different salt and light conditions.**

Root lengths were measured after 3-days-incubation on NaCl treatment plates. 20–30 roots in each plate were measured and standard deviations are presented next to the average length (mm).

strigolactone levels (Koltai et al., 2011) and strigolactone increases drought and salt tolerance of roots (Ha et al., 2014), is that the light-exposed roots are less sensitive to salt stress. Whereas the dark-grown roots show halotropism (salt avoidance tropism) by growing around the salt-enriched agar parts, the light-exposed roots grow into the salt-enriched agar if NaCl levels are not too high (**Tables 1** and **2**). As light-induced strigolactone levels modify also root responses to low phosphate (Mayzlish-Gati et al., 2012), one can propose that exposure of *Arabidopsis* roots to light is modifying their whole physiology. In fact, our preliminary data reveal that*Arabidopsis* roots exposed to light show changes the sensitivity also to the aluminum toxicity (Kagenishi et al., in preparation). In addition, IAA levels in light exposed (1 h) maize roots were more than doubled compared to control levels (Yokawa et al., in preparation). It is intriguing that enhanced strigolactone levels resemble light-induced effects on roots. It was shown that light impacts on the actin cytoskeleton, also increased abundance and recycling of PIN2 auxin transporter (Laxmi et al., 2008; Dyachok et al., 2011; Wan et al., 2012; Pandya-Kumar et al., 2014). Moreover, salt stress, drought stress, cold stress, alkaline stress, aluminum toxicity; all these challenges target the PIN2 auxin transporter which is expressed in the root apex transition zone (discussed in Baluška et al., 2010; Baluška and Mancuso, 2013). In order to avoid light exposure of roots, we recommend using of the partially darkened Petri dishes (Yokawa et al., 2011, 2013; Xu et al., 2013). This is important as stressing roots with light is modifying not only root responses to diverse stresses but also the overall physiology of such seedlings. For example, it has been reported that circadian rhythms in *Arabidopsis* roots are governed by the shoot part (James et al., 2008). Importantly in this respect, ROS signaling and homeostasis was shown to be a major modulator of circadian rhythms both in prokaryotes and eukaryotes (Edgar et al., 2012; Lai et al., 2012; Stangherlin and Reddy, 2013). It is possible that seedlings will change even their circadian clock when roots are illuminated.

Although plant roots are heterotrophic plant organs, they require active suppression of photosynthetic gene expression. Tyrosylprotein sulfotransferase (TPST) protein HPS7 emerged recently as crucial player controlling this active suppression (Kang et al., 2014). Interestingly enough, *hsp7* mutant roots show enhanced photosynthesis-related effects under phosphate deficiency stress. Previous studies identified Golden-Like transcription factors GLK1 and GLK2 involved in activation of photosynthetic genes in roots (Waters et al., 2009; Kobayashi et al., 2013). Both *hsp7* and GLK-ox mutant roots do not show stunted root growth and any other phenotypes when grown in darkness (Kang et al., 2014).

Finally, the plant physiology perspective of the light-induced root growth in *Arabidopsis* is considering the high root growth rate as a sign of optimal root growth conditions. In stark contrast, the plant neurobiology interpretation of this increased root growth rate is that the light-exposed roots are experiencing stress and that such stressed roots are trying to escape from this unfavorable situation (Yokawa et al., 2011, 2013; Xu et al., 2013). Alpi et al. (2007), critics of the plant neurobiology initiative claimed that '... plant neurobiology does not add to our understanding of plant physiology, plant cell biology or signaling.' Now, some 7 years later, it is getting obvious that plants and their roots are behaviorally much more complex than envisioned in the framework of classical plant physiology (Trewavas, 2005, 2009, 2014; Trewavas and Baluška, 2011; Baluška and Mancuso, 2013; Pierik and Testerink, 2014). Illumination of the roots is common laboratory praxis ever since *Arabidopsis thaliana* was introduced as model organism to plant sciences. However, it is important to be aware that light exposure of roots emerges as stress factor for the laboratory grown *Arabidopsis* seedlings. This is clear example for the usefulness of viewing plants as actively living and sensitive organisms solving their own plant-specific problems in intelligent manner.

### **ACKNOWLEDGMENTS**

Ken Yokawa was supported by the JSPS (Japanese Society for the Promotion of Science) Postdoctoral Fellowship. We thank Prof. Gareth I. Jenkins (University of Glasgow) for providing us with seeds of the UVR8::GFP line.

### **REFERENCES**

Darwin, C. R. (1880). *The Power of Movements in Plants*. London: John Murray.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 27 October 2014; accepted: 28 November 2014; published online: 12 December 2014.*

*Citation: Yokawa K, Fasano R, Kagenishi T and Baluška F (2014) Light as stress factor to plant roots – case of root halotropism. Front. Plant Sci. 5:718. doi: 10.3389/fpls. 2014.00718*

*This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science.*

*Copyright © 2014 Yokawa, Fasano, Kagenishi and Baluška. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Beneficial soil bacterium *Bacillus subtilis* (GB03) augments salt tolerance of white clover

*Qing-Qing Han1†, Xin-Pei Lü1†, Jiang-Ping Bai 2, Yan Qiao2, Paul W. Paré3, Suo-Min Wang1, Jin-Lin Zhang1 \*, Yong-Na Wu1, Xiao-Pan Pang1, Wen-Bo Xu1 and Zhi-Liang Wang1*

*1 State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, China 2 Gansu Key Laboratory of Crop Genetic and Germplasm Enhancement, Gansu Provincial Key Laboratory of Arid Land Crop Science, College of Agronomy, Gansu*

*Agricultural University, Lanzhou, China 3 Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA*

### *Edited by:*

*Vadim Volkov, London Metropolitan University, UK*

#### *Reviewed by:*

*Vadim Volkov, London Metropolitan University, UK Salman Gulzar, University of Karachi, Pakistan Yinglong Chen, The University of Western Australia, Australia*

#### *\*Correspondence:*

*Jin-Lin Zhang, State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, 768 West Jiayuguan Road, Chengguan District, Lanzhou 730020, Gansu, China e-mail<EMAIL_ADDRESS>
*†These authors have contributed equally to this work.*

Soil salinity is an increasingly serious problem worldwide that reduces agricultural output potential. Selected beneficial soil bacteria can promote plant growth and augment tolerance to biotic and abiotic stresses. *Bacillus subtilis* strain GB03 has been shown to confer growth promotion and abiotic stress tolerance in the model plant *Arabidopsis thaliana.* Here we examined the effect of this beneficial soil bacterium on salt tolerance in the legume forage crop, white clover. Plants of white clover (*Trifolium repens* L. cultivar Huia) were grown from seeds with or without soil inoculation of the beneficial soil bacterium *Bacillus subtilis* GB03 supplemented with 0, 50, 100, or 150 mM NaCl water into soil. Growth parameters, chlorophyll content, malondialdehyde (MDA) content and osmotic potential were monitored during the growth cycle. Endogenous Na+ and K+ contents were determined at the time of harvest. White clover plants grown in GB03-inoculated soil were significantly larger than non-inoculated controls with respect to shoot height, root length, plant biomass, leaf area and chlorophyll content; leaf MDA content under saline condition and leaf osmotic potential under severe salinity condition (150 mM NaCl) were significantly decreased. Furthermore, GB03 significantly decreased shoot and root Na+ accumulation and thereby improved K+/Na+ ratio when GB03-inoculated plants were grown under elevated salt conditions. The results indicate that soil inoculation with GB03 promotes white clover growth under both non-saline and saline conditions by directly or indirectly regulating plant chlorophyll content, leaf osmotic potential, cell membrane integrity and ion accumulation.

**Keywords:** *Bacillus subtilis***, white clover, salt tolerance, MDA, osmotic potential, Na+ accumulation**

### **INTRODUCTION**

Soil salinity has a significant negative impact on global agricultural productivity, and is a particularly acute issue in both irrigated and non-irrigated areas of the world (Flowers, 2004; Zhang et al., 2010a; Kronzucker and Britto, 2011; Zhang and Shi, 2013). Most crop and forage plants that feed the global population are sensitive to high salt concentration in soils (Rengasamy, 2010; Kronzucker et al., 2013). Soil salinity promotes osmotic stress, water deficit, stomatal closure and reduced leaf expansion (Rahnama et al., 2010; James et al., 2011); moreover, soil salinity causes deficiency of essential nutrients such as K+. Elevated Na+ inside plants can decrease plant photosynthetic rates and biomass accumulation (Mahajan and Tuteja, 2005; Munns and Tester, 2008; Zhang et al., 2010a; Zhang and Shi, 2013). Therefore, there is a need to find new ways to cope with the threat of global soil salinization to agriculture.

Beneficial interactions between bacteria and plants accelerate seed germination, promote growth, increase crop yields and secondary metabolites, and augment reproductive success.  *Bacillus subtilis* that is not toxic to humans widely exists in soils and can produce a wealth of antibacterial substances including lipopeptides, polypeptides, and phospholipids (Stein et al., 2002). Recently, Medeiros et al. (2011) found that transcriptional profiling in cotton was linked with *Bacillus subtilis* (UFLA285) which promoted biotic-stress tolerance. Zhang et al. (2011) found that a bioorganic fertilizer could effectively control banana wilt by strong colonization of *Bacillus subtilis* N11. *B. subtilis* strain GB03 that emits a complex blend of volatile organic compounds (VOCs) enhances growth and abiotic stress tolerance in *Arabidopsis* (Ryu et al., 2004; Farag et al., 2006). A bouquet of over 25 bacterial volatile odors have been identified that trigger differential expression of approximately 600 *Arabidopsis* transcripts related to cell wall modifications, primary and secondary metabolism, stress responses, hormone regulation and other expressed proteins (Zhang et al., 2007). In *Arabidopsis*, GB03 regulates auxin homeostasis and cell expansion (Zhang et al., 2007), augments photosynthesis by decreasing glucose sensing and ABA levels (Zhang et al., 2008b); promotes salt tolerance as well as reduces total Na+ by regulating tissue specific expression of *AtHKT1* (Zhang et al., 2008a). GB03 has also been shown to stimulate iron acquisition and increased photosynthetic capacity (Zhang et al., 2009). More recently GB03 has been found to improve osmotic-stress tolerance by elevating levels of endogenous osmoprotectants (Zhang et al., 2010b). With such beneficial plant responses activated by GB03, a legume forage crop is now being examined as to how it responds to this beneficial microbe.

White clover (*Trifolium repens* L.) is an important forage crop worldwide. Like many forage crops, white clover is sensitive to soil salinity (Rogers et al., 1997). Salt stress can decrease both above-ground growth and the number of root nodules that in turn compromise nitrogen fixation and soil fertility (Acharya et al., 2011). Since GB03 promotes growth and salt tolerance in *Arabidopsis*, the question of how GB03 improves growth and salt tolerance in the salt-sensitive forage crop white clover was addressed. The aim of current work was to evaluate the efficiency of *Bacillus subtilis* GB03 for growth promotion and salt tolerance in white clover.

### **MATERIALS AND METHODS**

### **BACTERIAL SUSPENSION CULTURE**

*Bacillus subtilis* strain GB03 was streaked onto LB agar plates and incubated at 28◦C without light for 24 h (The bacterium strain was presented by Professor Paul W. Paré at Texas Tech University, USA). Bacterial cells were then harvested from LB agar plates, transferred into liquid LB media and cultured at 28◦C with 250 rpm rotation to yield 109 colony forming units (CFU) mL−1, as determined by optical density and serial dilutions (Zhang et al., 2008a).

### **PLANT GROWTH AND TREATMENTS**

White clover (*Trifolium repens* L. cultivar Huia) seeds were surface sterilized with 2% NaClO for 1 min followed by 70% ethanol for 10 min, and rinsed with sterile water five times (The white clover seeds was presented by Dr. Wanhai Zhou at Gansu Agricultural University, China). Seeds were then sown in presterilized plastic pots (diameter 20 cm, depth 22 cm, 10 seeds/pot with eight replicates) containing 600 g of heat-sterilized (95◦C, 48 h) vermiculite and soil mix (1:1) watered with half strength Hoagland's nutrient solution (5 mM KNO3, 1 mM NH4H2PO4, 0.5 mM Ca(NO3)2, 0.5 mM MgSO4, 60µM Fe-citrate, 92µM H3BO3, 18µM MnCl2·4H2O, 1.6µM ZnSO4·7H2O, 0.6µM CuSO4·5H2O, and 0.7µM (NH4)6Mo7O24·4H2O). Each pot was watered with nutrient solution (400 mL) once per week. After germination, six uniform seedlings per pot were selected for continued growth and inoculated directly to the soil with 1 mL bacterial suspension culture as inoculation treatment or 1 mL liquid LB medium as a control. For salt stress treatments, seedlings were watered with 0, 50, 100, and 150 mM NaCl present in the nutrient solution. Each treatment contained eight pots as replications. Plants were grown in a glasshouse at the temperature regulated to around 28◦C during the day and 22◦C at night. Relative humidity averaged 65 and 75% for day and night periods, respectively, with natural photoperiod and light intensity.

### **PLANT BIOMASS AND PHYSIOLOGICAL MEASUREMENTS**

Sixty-day old plants were used for plant growth measurements and physiological index determination. Whole plant leaves were harvested to count leaf number per plant, measure leaf area using a leaf area meter (Epson Perfection 4870 Photo scanner, Epson America Inc., Long Beach, CA, USA) (Ma et al., 2012). Average area per leaf was calculated from leaf area per plant divided by leaf number per plant.

Plants were removed from the pots and roots were water rinsed to remove attached soil. Shoot height and root length (root depth) were measured by a ruler. Then, root and shoot were separated and blotted gently. Fresh weights were determined immediately and samples were oven dried at 80◦C for 2 day for dry weight measurements.

Leaf chlorophyll content was estimated according to Porra et al. (1989). Fresh leaf samples were ground thoroughly with 80% acetone in the dark and centrifuged at 9000 g for 10 min at 4◦C. Absorbance reading (UV-2102C Spectrophotometer, Unico Instrument Co., Ltd, Shanghai, China) at 645 and 663 nm for collected supernatant was used to estimate total chlorophyll content based on chlorophyll equations of Porra et al. (1989).

are means and bars indicate SDs (*n* = 8). Columns with different letters

To probe oxidative stress the biomarker malondialdehyde (MDA) was extracted and measured spectrophotometrically using a thiobarbituric acid (TBA) protocol (Bao et al., 2009). Absorbance was determined at 450, 532, and 600 nm using a UV spectrophotometer (UV-2102C, Unico Instrument Co., Ltd, Shanghai, China).

Leaf osmotic potential (s) was measured according to Ma et al. (2012). Fresh leaf samples were frozen in liquid nitrogen. Cell sap was collected by thawing slowly and then s was determined using a cryoscopic osmometer (Osmomat-030, Gonotec GmbH, Berlin, Germany) at 25◦C. The readings (mol·L−1) were used to calculate the solute potential (s) in MPa with the formula s = − The readings ×*R*× *T*, here *R* = 0.008314 MPa · L · mol−1 · K−1 and *T* = 298.8K(Ma et al., 2012).

### **ION ANALYSIS**

Na+ and K+ contents were measured according to the method described by Wang et al. (2007). Roots were washed twice for eight min in ice-cold 20 mM CaCl2 to exchange cell wall-bound K+ and Na+ and the shoots were rinsed in deionized water to remove surface salts. Root and shoot were separated and samples oven dried at 70◦C for 2 day. Na+ and K+ were extracted from dried plant tissues in 100 mM acetic acid at 90◦C for 2 h. Ion analysis was performed using an atomic absorption spectrophotometer (2655-00, Cole-Parmer Instrument Co., Vernon Hills, IL, USA).

### **DATA ANALYSIS**

Results of the growth, physiological index, ion contents and ion ratio were presented as means with standard deviations (*n* = 8).

### **RESULTS**

#### *BACILLUS SUBTILIS* **GB03 PROMOTED WHITE CLOVER GROWTH**

*Bacillus subtilis* GB03 enhanced both shoot height and root length of white clover under both non-saline conditions and salinity stress (**Figure 1** and **Supplementary Figure 1**). Shoot height was increased by 60.1% (*P* < 0.05) under non-saline conditions; compared to corresponding media control, GB03 significantly improved shoot height by 57.4% (*P* < 0.01), 33.7% (*P* < 0.05), and 95.6% (*P* < 0.01) at 50, 100, and 150 mM NaCl treatments, respectively (**Figure 1A**). Root length was increased by 23.9% (*P* < 0.05) under non-saline conditions; compared to corresponding media control, GB03 significantly improved root length by 28.0% (*P* < 0.01), 15.4% (*P* < 0.01), and 16.9% (*P* < 0.05) with 50, 100, and 150 mM NaCl treatments, respectively (**Figure 1B**).

GB03 increased plant biomass of white clover under both non-saline conditions and salinity stress (**Figure 2**). Shoot fresh weight was increased 4.1-fold (*P* < 0.05) under non-saline conditions; compared to corresponding media control, GB03 significantly improved shoot fresh weight by 5.5-fold (*P* < 0.05), 6.9-fold (*P* < 0.01), and 3.0-fold (*P* < 0.05) with 50, 100, and 150 mM NaCl treatment, respectively (**Figure 2A**). Shoot dry weight was increased by 4.0-fold (*P* < 0.05) under non-saline conditions; compared to corresponding media control, GB03 significantly improved shoot dry weight by 4.9-fold (*P* < 0.05), 6.4-fold (*P* < 0.01), and 3.2-fold (*P* < 0.05) with 50, 100, and

dry weight. Values are means and bars indicate SDs (*n* = 8). Columns with different letters indicate significant difference at *P* < 0.05 (Duncan test).

150 mM NaCl treatment, respectively (**Figure 2B**). Root fresh weight was increased by 55.0% (*P* < 0.05) under non-saline conditions; compared to corresponding media control, GB03 significantly improved shoot dry weight by 44.1% (*P* < 0.05) and 27.7% (*P* < 0.05) at 50 and 100 mM NaCl treatment, respectively (**Figure 2C**). Root dry weight was increased by 74.7% (*P* < 0.01) under non-saline conditions; compared to corresponding media control, GB03 significantly improved shoot dry weight by 51.7% (*P* < 0.01) and 32.5% (*P* < 0.05) at 50 and 100 mM NaCl treatment, respectively (**Figure 2D**). GB03 had no significant effects on root fresh and dry weights under 150 mM NaCl stress.

### **GB03 INCREASED LEAF AREA AND CHLOROPHYLL CONTENT**

GB03 promoted leaf growth under both non-saline and salinity stress conditions (**Figure 3**). Leaf area per plant was increased by 3.1-fold (*P* < 0.05) under non-saline conditions; compared to corresponding media control, GB03 significantly increased leaf area per plant by 5.7-fold (*P* < 0.05), 7.8-fold (*P* < 0.01), and 1.5-fold (*P* < 0.05) with 50, 100, and 150 mM NaCl treatment, respectively (**Figure 3A**). Leaf number per plant was increased by 84.2 % (*P* < 0.05) under non-saline conditions; compared to corresponding media control, GB03 significantly elevated leaf number per plant by 142.1% (*P* < 0.05) and 107.7% (*P* < 0.05) with 50 and 100 mM NaCl treatments, respectively (**Figure 3B**). Average area per leaf was increased by 125.6% (*P* < 0.05) under non-saline conditions; compared to corresponding media control, GB03 significantly improved average area per leaf by 159.4% (*P* < 0.01), 334.5% (*P* < 0.01), and 81.5% (*P* < 0.01) with 50, 100, and 150 mM NaCl treatments, respectively (**Figure 3C**).

In addition to promoting leaf growth, *Bacillus subtilis* GB03 increased leaf chlorophyll content under both non-saline and salinity stress (**Figure 4**). Leaf chlorophyll content rose by 36.0% (*P* < 0.01) under non-saline condition; compared to corresponding media control, GB03 significantly increased leaf chlorophyll content by 34.3% (*P* < 0.01), 37.5% (*P* < 0.01), and 57.4% (*P* < 0.01) with 50, 100, and 150 mM NaCl treatments, respectively (**Figure 4**).

### **GB03 REDUCED LEAF OSMOTIC POTENTIAL UNDER HIGHER SALINITY CONDITION**

At lower salt concentration (0, 50, and 100 mM NaCl), compared with the controls, GB03 had no significant effect on leaf osmotic potential; however, under higher salt concentration (150 mM NaCl), GB03 significantly decreased leaf osmotic potential of white clover by 46.7% (*P* < 0.05) (**Figure 5**).

### **GB03 REDUCED LEAF MALONDIALDEHYDE (MDA) CONTENT UNDER SALINITY CONDITIONS**

The results indicated that GB03 reduced leaf MDA content under both non-saline conditions and salinity stress (**Figure 6**). Leaf MDA content was not changed under non-saline conditions; however, compared to corresponding media control, GB03 significantly reduced leaf MDA content by 52.7% (*P* < 0.01), 56.8% (*P* < 0.01), and 53.8% (*P* < 0.05) with 50, 100, and 150 mM NaCl treatments, respectively (**Figure 6**), thus, oxidative stress tolerance and integrity of the cell membrane as measured by the biomarker MDA were both favorably regulated by GB03.

**FIGURE 3 | Effects of GB03 bacterization on leaf development and growth of white clover under various concentrations of NaCl. (A)** leaf area per plant, **(B)** leaf number per plant, and **(C)** average area per leaf. Values are means and bars indicate SDs (*n* = 8). Columns with different letters indicate significant difference at *P* < 0.05 (Duncan test).

### **GB03 REDUCED SODIUM ACCUMULATION UNDER SALINITY CONDITIONS**

To test whether GB03 inoculation resulted in altered ion accumulation in white clover under various salinity conditions, endogenous Na+ and K+ content was measured in the 0, 50, 100, and 150 mM NaCl treatments. K+ accumulation was uneffected by GB03 inoculation (**Figures 7A,B**). However, endogenous Na+ accumulation was reduced significantly. Compared to the media control, GB03 significantly decreased shoot Na+ content by 40.7% (*P* < 0.01), 22.5% (*P* < 0.01), and 26.3% (*P* < 0.01), as well as root Na+ content by 27.1% (*P* < 0.05), 39.7% (*P* < 0.01), and 40.7% (*P* < 0.01) with 50, 100, and 150 mM NaCl treatments, respectively (**Figures 7C,D**).

K+/Na+ ratios were also significantly improved by GB03 inoculation with 50, 100, and 150 mM NaCl treatments. Compared to corresponding media control, Shoot K+/Na+ ratios increased by 78.6% (*P* < 0.01), 32.0% (*P* < 0.05), and 39.7% (*P* < 0.05) as well as root K+/Na+ ratios by 34.6% (*P* < 0.05), 82.2% (*P* < 0.01), and 66.2% (*P* < 0.01) under soil salinity conditions of 50, 100, and 150 mM NaCl, respectively (**Table 1**).

### **DISCUSSION**

### **INFLUENCE OF** *BACILLUS SUBTILIS* **GB03 ON GROWTH OF WHITE CLOVER UNDER SALINE CONDITION**

Beneficial soil bacteria promoted plant growth of many plant species (Bashan et al., 2000; Gray and Smith, 2005; Xie et al., 2009; Paré et al., 2011). Under salinity stress, inducible plant growth promotion mediated by beneficial soil bacteria has also been observed in several cultivated and wild plant species including dwarf saltwort (*Salicornia bigelovii*) (Bashan et al., 2000), tomato

(*Lycopersicon esculentum*) (Mayak et al., 2004), chickpea (*Cicer arietinum*) (Mhadhbi et al., 2004), alfalfa (*Medicago sativa*) (Ibragimova et al., 2006), common glasswort (*Salicornia europea*) (Ozawa et al., 2007), maize (*Zea mays*) (Bano and Fatima, 2009), and wheat (*Triticum aestivum*) (Tiwari et al., 2011). The growth promotion of white clover by GB03 inoculation in soil evaluated in the present study is consistent with previous reports in *Arabidopsis* (Zhang et al., 2007, 2008a,b, 2010b; Xie et al., 2009; Paré et al., 2011). Interestingly, our results indicated that GB03 is a more efficient promoter of shoot growth than root growth in white clover, especially under salt exposure (50, 100, and 150 mM NaCl). Whether GB03 promotes growth of white clover by enhancing nitrogen fixation and the coordination between nitrogen-fixing bacteria and *Bacillus subtilis* (GB03) in the rhizosphere remain to be investigated.

Leaf development plays an important role in plant production since it affects the area available for photosynthesis, which is strongly related to plant growth and biomass accumulation (Gutierrez-Boem and Thomas, 1998; Battie-Laclau et al., 2013). In our study, GB03 inoculation significantly increased leaf area per plant, leaf number per plant, and thereby average area per leaf in white clover. Leaf chlorophyll content is also an important physiological trait directly linked to photosynthesis rate in plants (Ma et al., 2012). Previous studies showed that plants grown under salinity conditions produced less chlorophyll and dry matter than those without salinity stress due to chlorophyll peroxidation (Hernandez et al., 1995; Zayed and Zeid, 1998; Lunde et al., 2007; Tuna et al., 2008; Barry, 2009). Zhang et al. (2008b) found that GB03 augments photosynthetic capacity by increasing photosynthetic efficiency and chlorophyll content in *Arabidopsis*. With white clover, GB03 significantly improved leaf chlorophyll content under both non-saline and salinity conditions (50, 100, and 150 mM NaCl).

### **GB03 AMELIORATED LEAF OSMOTIC ADJUSTMENT ABILITY AND ALLEVIATED CELL MEMBRANE DAMAGE UNDER SALINE CONDITIONS**

When plants are exposed to saline or drought conditions, osmotic stress rapidly follows (Munns and Tester, 2008). Salt stress reduces

**Table 1 | Effects of GB03 bacterization on K+/Na+ ratio in shoot and root of white clover under various concentrations of NaCl.**

*Values are means* ± *SDs (n* = *8). Different letters indicate significant difference at P* < *0.05 (Duncan test) within shoots and roots, individually.*

the rates of photosynthesis and transpiration, stomatal conductance and leaf area etc. (Wang et al., 2005; Ramani et al., 2006). Leaves could accumulate abundant osmoprotectant and adjust their osmotic potential (≈leaf water potential) below that of the apoplast and soil in order to ensure that the plants can continue to absorb moisture from the soil and maintain turgor pressure, thus improving their stress tolerance (Bao et al., 2009; Jha et al., 2011; Janz and Polle, 2012; Ma et al., 2012).

Zhang et al. (2010b) reported that GB03 enhanced *Arabidopsis* choline and glycine betaine synthesis associated with enhanced osmolyte content, resulting in increased plant tolerance to osmotic stress. In the case of white clover, GB03 significantly reduced leaf osmotic potential under severe salt stress conditions (150 mM NaCl).

Soil salinity is known to increase the level of reactive oxygen species in plant leaves, which are well recognized for membrane lipid peroxidation and cause an increase in leaf malondialdehyde (MDA), a product of membrane lipid peroxidation (Koca et al., 2006; Yazici et al., 2007). Therefore, leaf MDA content, representing the degree of cell membrane damage, is usually used to evaluate plant tolerance to salinity and drought (Luna et al., 2000; Miao et al., 2010). In this work, GB03 significantly decreased leaf MDA content of white clover under saline conditions (50, 100, and 150 mM NaCl).

### **GB03 REDUCED Na+ CONTENT IN PLANTS**

(Duncan test).

Saline soils contain a varied and complex array of cation-anion pairs (e.g., Na2SO4, MgSO4, CaSO4, MgCl2, KCl, and Na2CO3), with Na+ often the dominant species (Zhang et al., 2010a). Growth inhibition, a common plant response to soil salinity, is correlated with high internal Na+ concentration and low K+/Na+ ratio in the plant (Zhang et al., 2010a). Plants grown in saline conditions can minimize Na+ toxicity by restricting Na+ uptake and Na+ xylem loading, extruding Na+ from root cells and redirecting Na+ from shoots to roots (Tester and Davenport, 2003; Munns and Tester, 2008; Zhang et al., 2010a; Kronzucker and Britto, 2011; Zhang and Shi, 2013). Zhang et al. (2008a) found that in *Arabidopsis* GB03 decreased whole plant Na+ content to 54% of that in control plants by down-regulating *HKT1* expression in roots to decrease root Na+ uptake and up-regulating *HKT1* expression in shoots to enhance shoot-to-root Na+ recirculation, respectively. In this study, GB03 soil inoculation significantly decreased Na+ accumulation and increased K+/Na+ in both shoots and roots of white clover under salt stress with no measurable effect on K+ content in the whole plant. Whether GB03 improves salt tolerance of white clover by tissue specific regulation of *HKT* gene expression remains to be investigated.

In summary, the results presented here established that the inoculation of the soil bacterium *Bacillus subtilis* GB03 to the rhizosphere significantly increases plant growth and biomass of the forage legume white clover under both non-saline and saline conditions. GB03-regulated plant processes include chlorophyll abundance, leaf osmotic potential, cell membrane integrity, and ion accumulation. This study provides insight for the application of selected bacteria to cultivated legumes to combat saline toxicity.

### **ACKNOWLEDGMENTS**

This research was supported by the National Basic Research Program of China (grant No. 2014CB138701), the National Natural Science Foundation of China (grant No. 31172256, 31222053), the Programs for New Century Excellent Talents, Ministry of Education, China (grant No. NCET-11-0217) and the Fundamental Research Funds for the Central Universities (grant No. LZUJBKY-2013-K08, BT05, and 2014-M01).

### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fpls.2014. 00525/abstract

#### **Supplementary Figure 1 | Effects of bacterization on growth of white**

**clover plants in pots under various concentrations of NaCl.** Here GB03 represents *Bacillus subtilis* GB03 suspension culture in LB medium, LB Luria Broth medium without bacteria and 0, 50, 100, and 150 the concentrations of NaCl (mM).

### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 19 August 2014; accepted: 16 September 2014; published online: 08 October 2014.*

*Citation: Han Q-Q, Lü X-P, Bai J-P, Qiao Y, Paré PW, Wang S-M, Zhang J-L, Wu Y-N, Pang X-P, Xu W-B and Wang Z-L (2014) Beneficial soil bacterium Bacillus subtilis (GB03) augments salt tolerance of white clover. Front. Plant Sci. 5:525. doi: 10.3389/ fpls.2014.00525*

*This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science.*

*Copyright © 2014 Han, Lü, Bai, Qiao, Paré, Wang, Zhang, Wu, Pang, Xu and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*


**Acknowledgement**

362 Chromatography – The Most Versatile Method of Chemical Analysis

**1798 20**

**6. Conclusions**

micelles)

predictive power.

**Author details**

Mihalj Poša

• Critical Micellar Concentration • Haematolityc potentitial • Binding for Albumin

• Solubilization of cholesterol, lecitin, vitamin E etc.

acids:

*score* **PC2**

**5**

**15 16**

**7**

**10**

**21**

**I**

**Figure 14.** Grouping of bile acids (Table 4) based on the scores of principal component.

**6**

**23**

*LR*

**22**

**24 25** This study was supported by the Secretariat for Science and Technological Development of AP Vojvodina, Republic of Serbia (Grant No.<PHONE_NUMBER>/2012).