Protoplasma DOI 10.1007/s00709-014-0669-1
ORIGINAL ARTICLE
Apoplastic barrier development and water transport in Zea mays seedling roots under salt and osmotic stresses Jie Shen & Guoxin Xu & Hui Qiong Zheng
Received: 12 September 2013 / Accepted: 16 June 2014 # Springer-Verlag Wien 2014
Abstract The development of apoplastic barriers was studied in Zea mays seedling roots grown in hydroculture solution supplemented with 0–200 mM NaCl or 20 % polyethylene glycol (PEG). Casparian bands in the endodermis of both NaCl- and PEG-treated roots were observed closer to the root tip in comparison with those of control roots, but the cell wall modifications in the endodermis and exodermis induced by salt and osmotic stresses differed. High salinity induced the formation of a multiseriate exodermis, which ranged from several cell layers to the entire cortex tissue but did not noticeably influence cell wall suberization in the endodermis. In contrast, osmotic stress accelerated suberization in both the endodermis and exodermis, but the exodermis induced by osmotic stress was limited to several cell layers in the outer cortex adjacent to the epidermis. The hydrostatic hydraulic conductivity (Lp) had decreased significantly after 1 day of PEG treatment, whereas in NaCl-treated roots, Lp decreased to a similar level after 5 days of treatment. Peroxidase activity in the roots increased significantly in response to NaCl and PEG treatments. These data indicate that salt stress and osmotic stress have different effects on the development of apoplastic barriers and water transport in Z. mays seedling roots.
Keywords Apoplastic barriers . Osmotic stress . Salt stress . Water transportation . Zea mays
Handling Editor: Néstor Carrillo Electronic supplementary material The online version of this article (doi:10.1007/s00709-014-0669-1) contains supplementary material, which is available to authorized users. J. Shen : G. Xu : H. Q. Zheng (*) Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, 200032 Shanghai, China e-mail: [email protected]
Introduction Roots of all vascular plants possess an endodermis, which occupies an interfacial position between ion absorbing and transporting tissue and provides a barrier to the apoplast of ions and other solutes (Bonnett 1967; Peterson et al. 1993; Naseer et al. 2012; Lee et al. 2013). According to Kroemer (1903) followed by Robards and Jackson (1975) and Geldner (2013), four developmental stages of the endodermis can be distinguished: (1) the pro-endodermis, (2) the primary stage endodermis with the Casparian band, (3) the secondary stage endodermis with the deposition of suberin lamellae on the internal wall surface, and (4) the tertiary stage endodermis with heavy U-shaped cell wall deposition on the inner tangential and the radial cell walls of the endodermal cells. Apoplastic transport in plant roots is blocked when they form Casparian strips (Schreiber et al. 1999; Ranathunge and Schreiber 2011; Halpin 2013). Experimental injuring of the endodermis indicates that the Casparian band has little effect on the movement of water, but it largely prevents the movement of ions, and the main resistance to water movement lies in the living tissues of the root (Peterson and Steudle 1993). In addition to an endodermis, many plants develop an exodermis, which is the outermost layer(s) of the cortex and undergoes cell wall modifications with the deposition of Casparian bands and suberin lamellae. Plant response to environmental stimuli by modifying the degree of suberization and lignification of root cell walls has been extensively studied (Negrel et al 1993; Bolwell et al. 1997; Franke and Schreiber 2007; Bernards and Lewis 1998; Geldner 2013; Halpin 2013). Apoplastic barriers, which are composed of suberin and restrict water and nutrient loss and prevent the invasion of pathogens, were strengthened by various stress conditions (Höfer et al. 2008; Krishnamurthy et al. 2009). The amounts of endodermal suberin and lignin increase 1.5-fold and 2.0-fold, respectively, in corn roots
S. Jie et al.
treated with NaCl and polyethylene glycol (PEG) (Schreiber et al. 1999). Thomas et al. (2007) reported strong negative correlations between the amount of the aliphatic component of root suberin and plant mortality induced by Phytophthora sojae. These modifications of cell walls lead to a decrease in radial water transport in the roots (reviewed by Hose et al. 2001; Schreiber et al. 2005; Franke and Schreiber 2007), such as the exodermis, which increases resistance to radial water movement, making it more difficult for the root to take up water (Peterson and Enstone 1996). The physiological importance of apoplastic barriers in many plants is well known (Zimmermann et al. 2000; Schreiber et al. 2005; Naseer et al. 2012; Lee et al. 2013). However, how environmental factors influence the formation of apoplastic barriers remains enigmatic. Deeper insight into the development of apoplastic barriers is required to elucidate the factors that determine barrier properties. In the present research, we studied exodermal and endodermal development in relation to the formation of apoplastic barriers in growing Zea mays roots under salt stress and osmotic stress conditions, respectively. Our data indicated that the properties of apoplastic barriers in the root are tissue specific and regulated in response to different environmental stress conditions.
Materials and methods Plant material Seeds of maize (Z. mays L. cv. Suyu NO.1) were surface sterilized with a solution of sodium hypochlorite (0.5 %, w/ v) and Tween 20 (0.75 %, w/v) for 10 min. After thorough rinsing with distilled water, the seeds were imbibed and germinated on filter paper moistened with distilled water for 2 days at 26–27 °C in the dark. Seedlings with roots about 3 cm long were chosen and grown in culture chambers containing 5-L Hoagland nutrient hydroculture solution (Hoagland and Arnon 1938). The control consisted of basic Hoagland solution. Salt and drought treatments comprised the basic nutrient solution supplemented with 100–200 mM NaCl and 20 % PEG 6000 (w/v), respectively. The total volume of the solution in each culture chamber was kept constant by adding double-distilled water to compensate for water loss through plant evapotranspiration. The osmotic potential of the culture solutions was measured with a vapor pressure osmometer (Wescor, Logan, UT, USA). The seedlings were grown in a greenhouse (26–27 °C) with a 16-h photoperiod at 200 μmol m−2 s−1 photon flux rate and harvested 8–12 days after initiation of treatment. Histochemical staining for light microscopy The development of root endodermal and exodermal cells was examined with a series of freehand sections. Casparian bands
were detected by staining with the fluorescent dye neutral red and toluidine blue, or berberine hemisulfate, as described by Brundrett et al. (1988), and suberin lamellae were detected by staining with Sudan III, as described by Schreiber et al (1999). Measurement of hydraulic conductivity Root hydrostatic hydraulic conductivity (Lp) was measured in accordance with the method of Miyamoto et al. (2001). Briefly, roots were enclosed in a steel chamber, and pneumatic pressure was applied to the root bath medium (water). The pressure in the chamber (Pgas) was raised in steps of 0.05 up to 0.3 MPa above atmospheric pressure. The exuded sap was collected, and the volume of the exuded sap (Jv; in m3 m−2 s−1) was calculated. The value of Lp was calculated according to the formula: . Lp ¼ J v Pgas þ σ•ðΠ b −Π s Þ
where σ is the root reflection coefficient for nutrient salts in the xylem, which was estimated to be σ=0.4, and Πb and Πs were the osmotic pressure of the bath medium and sap exuded from individual plants, respectively. At a high value of Pgas (≥0.1 MPa), the effect of the osmotic term is not pronounced, so σ, Πb, and Πs can be ignored (Miyamoto et al. 2001). Lp was determined from plots of Jv against Pgas. Histochemical localization of peroxidase activity Peroxidase (POD) activity was monitored as described by Ros-Barcelo et al. (2002) with modifications. Root sections were incubated for 20 min at 25 °C in a staining solution composed of 0.1 mg mL−1 tetramethylbenzidine (TMB)-HCl in 50 mM Tris-acetate buffer (pH 5.0). Staining was monitored in 25 sections selected randomly from 20 to 30 roots.
Results Growth and development of primary roots under salt and osmotic stresses Both salinity and drought stresses result in decreased availability of water to plant cells and affect plant growth and development. To understand how Z. mays plants changed their growth and development to counteract the decreased availability of water under these stresses, we treated seedlings with low water potential (ψw) by using nutrient solutions supplemented with serial concentrations of NaCl or 20 % PEG 6000. After culture in nutrient solution containing 50, 100, 150, or 200 mM NaCl for 8–12 days, the treated seedlings exhibited
Apoplastic barrier development and water transport in Zea mays
obvious stress symptoms but survived up to 200-mM NaCl treatment (Fig. S1). The total root length and the sites of the first lateral root and root hair development were significantly affected by NaCl at concentrations exceeding 100 mM (Fig. 1, Table S1). The total length of primary roots under the NaCl- or PEG-treated conditions relative to that under the control condition was significantly reduced by 47 and 62 %, respectively (Table 1). The length of the elongation region of 100 mM NaCl- or 20 % PEG-treated roots was reduced by 80 and 88 %, respectively, whereas the size of root caps showed no significant difference. The distance from the site of the first lateral root to the root apex under the NaCl- or PEG-treated conditions was reduced by 83 and 51 %, respectively. The distance from the site of the first lateral root to the root apex under NaCl stress was about 3-fold shorter than that under PEG stress. In contrast, the distance from the site of root hair development to the root apex in PEG-treated roots was 1.7-fold lower than that in NaCl-treated roots (Table 1). These results indicate that young maize seedlings respond differentially to salt and drought stress. Hydraulic conductivity of seedling roots under salt and osmotic stresses The Lp value was not significantly affected by 100-mM NaCl treatment after 1 day but was reduced by 34.5 % after treatment for 5 days compared with that in the control roots. The Lp values in 20 % PEG-treated roots decreased by 69 and 93 % after 1 and 5 days of treatment, respectively, compared with those in the control roots (Fig. 2). These data showed that PEG treatment decreased Lp within a short period (less than 1 day). Treatment with NaCl also affected the Lp value in the roots but only after a longer period in comparison with PEG treatment. Development of endodermis and exodermis under salt and osmotic stresses Previous studies indicate that the apoplasmic water permeability might depend on the developmental state of the root (Peterson et al. 1993). To evaluate how the hydraulic resistance of the root changes in response to external stresses (e.g., drought and salinity), the formation of apoplastic barriers (Casparian bands and suberin lamellae) in the endodermis and exodermis under osmotic and salinity stresses in young roots was examined. The roots of seedlings grown under NaCl and PEG treatments and the control conditions were fixed, and cross sections about 0.1-mm thick were cut from the root tips continuously. Each section was examined with an epifluorescence microscope to observe the cell wall modifications (Fig. S2). When the cells of an 8-day-old seedling grown under the control condition were arranged in sequence basipetally from the root tip, the endodermal cells were observed to
Fig. 1 Phenotype of Zea mays seedlings (8 days old) cultured under the control condition, 100 mM NaCl condition, and 20 % PEG condition, respectively. Bar 5 cm
be at the primary stage with the Casparian strip present in the region about 13–38 mm from the root tip (Fig. 3a). Endodermal cells at the secondary stage, showing deposition of suberin lamellae on the internal wall surface, were observed in the region about 38–70 mm from the root tip (Figs. 3b and 4). About 95–110 mm from the root apex, the endodermal cells displayed a strong U-shaped cell wall deposition on the inner tangential and the radial cell walls and had developed into the tertiary stage (Fig. 3c). The sites of endodermal Casparian bands were observed closer to the root tip in both NaCl- and PEG-treated roots in comparison with those under the control condition. The distance from the root tip to the site of the first lateral root in control roots was about 70 mm but was decreased to about 11 and 34 mm in 100-mM NaCl- and 20 % PEG-treated roots, respectively. The Casparian strip had developed into the tertiary stage at about 37 mm from the apex of PEG-treated roots compared with about 95 mm in control roots and about 85 mm in the NaCl-treated roots (Fig. 4). Suberin lamellae, corresponding to the Casparian strip, were detected in the endodermal cell walls of salt- and osmoticstressed roots and control roots by staining with Sudan III, a lipophilic dye commonly used to visualize suberin deposition. Compared with those in control roots (Fig. 5a), the Casparian strip in NaCl-treated roots showed decreased staining, whereas PEG-treated roots exhibited a distinct increase in staining intensity (Fig. 5b, c). This result suggests that suberin deposition in the cell wall during development of endodermal cells might be differentially modified by salt and osmotic stresses. The cross sections with secondary stage endodermis were selected for staining with the berberine–aniline blue procedure, which is a rapid and precise method for visualizing lignified cell walls, Casparian bands, suberin lamellae, and callose (Brundrett et al. 1988). When the sections from control
S. Jie et al. Table 1 Different effect of salt and osmotic stress on the development of Zea mays seedling roots (mean±SD, n=200) Treatment
Total lengtha (cm)
Root tip lengthb (cm)
Root cap size (μm)
First lateral rootc (cm)
Control 100 mM NaCl 20 % PEG
21.68±5.1 11.52±1.36 8.2±1.19
1.28±0.32 0.26±0.16 0.15±0.02
418±96 398±28 335±30
6.85±2.68 1.14±0.67 3.35±0.83
a
Total length of root
b
Distance from the root apex to the site of root hair appearance
c
Distance from the root apex to the site of the first lateral root appearance
roots were examined under ultraviolet light, tracheary element walls fluoresced intense yellow-white, the endodermal Casparian band fluoresced yellow-green, and cortical cells fluoresced blue (Fig. 6a). Staining of the endodermal Casparian bands in NaCl-treated roots was not noticeably different from those in control roots (Fig. 6a, b), whereas almost a full ring of endodermal cells with suberin lamellae was observed in PEG-treated roots (Fig. 6c). Intense yellowish green fluorescence was observed in all cell walls of parenchyma cells in the central and outer cortex of NaCl-treated roots (Figs. 6b and S3). A high concentration of NaCl promoted formation of exodermis in the cortex from several cell layers to the whole tissue (Figs. S3 and S4). This multiseriate exodermis also formed in PEG-treated roots but was limited to three to four layers of the outer cortical cells and never extended to the whole tissue (Fig. 6c). Peroxidase activity of seedling roots under salt and osmotic stresses Histochemical localization of POD by staining with the TMB reagent showed that POD activity in the primary root was strongly affected by treatment with 100 mM NaCl and 20 %
PEG. The activity of POD in the cell walls of the endodermis and vascular cylinder of both NaCl- and PEG-treated roots was visibly increased (Fig. 7b, c) in comparison with that of the control condition (Fig. 7a). In contrast, the activity of POD in the cell wall of epidermal cells increased under PEG treatment (Fig. 7c) but showed no obvious change under NaCl treatment (Fig. 7b) in comparison with that under the control condition (Fig. 7a). A recent study indicated that PODs used localized superoxide-derived H2O2 for Casparian strip formation (Lee et al. 2013). To test whether the production of apoplastic H2O2 in the endodermis is affected by NaCl and PEG treatment, we used the cerium chloride assay, in which cerium chloride forms electron-dense precipitates in the presence of H2O2 through formation of cerium perhydroxide. With this assay, we observed apparently localized cerium precipitations at the position of Casparian strips of endodermal cells in NaCl- and PEGtreated roots in comparison with that of control roots (Figs. S5 and S6). Cerium precipitations were also observed at the position of inner cell wall of epidermal cells in PEG-treated roots (Fig. S7f, i) but not in the NaCl-treated and the control roots (Fig. S7d, e, g, h). These results indicated that distribution of apoplastic H2O2 in the endodermis and exodermis was also differentially affected by NaCl and PEG treatment.
Discussion
Fig. 2 Effects of salt and osmotic stress on the hydraulic conductivity (Lp) of Zea mays seedling roots. Con control, NaCl 100-mM NaCl treatment, PEG 20 % PEG treatment
Several previous studies suggest that rapid responses (within a few hours of application) to salt often resemble responses to low ψw imposed using nonionic solutes (e.g., PEG 6000), whereas longer-term responses (from days to weeks) are more salt specific (Carillo et al. 2011; Munns 2002, 2011; Verslues et al. 2006). At the cellular level, the difference in tissue responses to isotonic salinity and osmotic treatments is also observed at the minute timescales, both in root (Chen et al. 2005) and shoot (Shabala 2000) tissues. The most pronounced structural responses of plants to salinity and osmotic stresses are often related to cell wall modification. Some fundamental
Apoplastic barrier development and water transport in Zea mays
Fig. 3 The Casparian strip in a Zea mays root stained with neutral and toluidine blue and viewed under UV light with an epifluorescence microscope. a, b, c Florescence micrographs showing endodermal
Casparian bands. d Diagram of a Zea mays seedlings, and the marked regions are correspondingly shown in a, b, c. X xylem, En endodermal cells, Co cortex cells. Bars 50 μm in a, b, and c
information regarding cell wall modification under stress conditions has been known for a long time. For example, salinity accelerates development of endodermis and induces formation of uniseriate, biseriate, or multiseriate exodermis in roots of many plant seedlings including monocots and dicots and members of primitive and advanced plant families (Reinhardt and Rost 1995; Degenhardt and Gimmler 2000; Karahara et al. 2004). However, it is only recently that significant new insights regarding the underlying molecular factors for localized lignin deposition in the endodermis have come to light (Lee et al. 2013). We are still largely ignorant about how plants use this mechanism of localized cell wall modification to cope with different stresses. Our results showed that the cell wall modifications in the developing endodermis of Z. mays seedling roots were affected by both NaCl and PEG treatment, but the effects of salt (NaCl) and osmotic (PEG) stress on the cell wall modifications of endodermal and exodermal cells differed markedly. Initiation of the Casparian strip was shown to be correlated with development of the phloem and xylem
(Wilcox 1954; Martinka et al. 2012), where root hairs develop from epidermal cells. The investigation by Haas and Carothers (1975) suggests that in Z. mays, there is a single precisely determined site at which Casparian strip formation occurs and that there is no prior or subsequent shift in this position. Our present results showed that the site of root hair development was closer to the root apex in PEG-treated roots than that in NaCl-treated roots. Comparisons of the development of endodermal cells in NaCl- and PEG-treated roots indicated that suberization in the endodermis was more strongly promoted by osmotic stress than that by salt stress (Fig. 6). Exposure to salinity induced apparent suberization of the cell wall from one cell layer to the entire cortex tissue with increasing concentration of NaCl, whereas osmotic stress induced formation of an exodermis limited to three to four layers of the outermost cortical cells. These results suggest that roots might adopt different mechanisms for the development of the Casparian strip and the deposition of suberin lamellae in the exodermis and endodermis to counteract salt and drought stresses. NaCl salinity induced suberization of the exodermis before suberization of the Casparian strip in the endodermis, whereas PEGinduced osmotic stress caused intensified suberization of exodermal and endodermal cell walls almost simultaneously. Salinity and drought cause intensified suberization in exodermal cell walls of several plant species (e.g., cotton, citrus, and sorghum) (Walker et al. 1984; Cruz et al. 1992; Reinhardt and Rost 1995; Simone et al. 2003), but roots that develop different strategies to cope with different stresses have not been reported. In the present study, no exodermis was observed at 95 mm from the root apex in control roots. However, in salt (100 mM NaCl)-treated roots, the exodermis was observed as close as 30–40 mm from the root apex, which coincides with the region that root hairs and lateral roots begin to develop. These sites will be critical for uptake of mineral nutrients and water by the root. When roots were treated with 200 mM NaCl, suberization of the exodermis was observed almost from the beginning of the region of root hair development. We hypothesize that the presence of a multilayer
Fig. 4 Distance from the root apex to the point of the endodermal Casparian strip appearance (stage A) and the endodermal suberin lamellae appearance in secondary stage (stage B) and in tertiary stage (stage C). Con control, NaCl 100 mM NaCl, PEG 20 % PEG
S. Jie et al. Fig. 5 Cross sections were made at the site of the first lateral root appearance and stained with Sudan III. The roots were grown under control (a), 100 mM NaCl (b), and 20 % PEG (c) conditions, respectively. En endodermal cells, X xylem. Bars 50 μm in a, b, and c
Fig. 6 Cross sections were made at the site of the first lateral root appearance and stained with berberine hemisulfate and aniline blue to illustrate the effects of salt and osmotic stresses on exodermal and endodermal development. a Control. b 100mM NaCl treatment. c 20 % PEG treatment. Co cortex, X xylem, En endodermal cells, Ep epidermal cells. Bars 100 μm in a, b, and c
exodermis in the outer cortex protects the inner cortex and vascular cells from exposure to the high ion concentration in the external solution; thus, suberization of the endodermis was not significantly accelerated in NaCl-treated roots compared with that in PEG-treated roots. The process of water movement through the root and the location of the major barrier(s) to water movement are still subjects of debate. The Casparian band, of which components are hydrophobic, has been assumed to be a barrier of radial Fig. 7 Histochemical localization of peroxidase in cross sections at the region of the first lateral root appearance, stained with the TMB reagent. a Control. b 100 mM NaCl. c 20 % PEG. Co cortex, X xylem, En endodermal cells, Ep epidermal cells. Bars 50 μm in a, b, and c
movement of water in the root (Frensch et al. 1996). However, Peterson et al. (1993) argued that the endodermis (with Casparian bands only) was not a major barrier to water movement into the root by determining the changes in hydraulic conductivity after injuring the endodermis (Peterson et al. 1993; reviewed by Steudle and Peterson 1998). One consistent observation regarding the radial cell-to-cell flow of water in the root is that suberin lamellae are barriers to water movement. Formation of an exodermis with suberin lamellae may
Apoplastic barrier development and water transport in Zea mays
add to the overall resistance to water flow. An exodermis is usually absent in young maize roots grown in hydroponics (Steudle and Peterson 1998; Zimmermann and Steudle 1998) and is induced by NaCl and PEG stress treatments. In the present study, the hydraulic conductivity (Lp) in both NaCland PEG-treated roots was significantly decreased, but Lp decreased more rapidly in response to PEG treatment than was observed in NaCl-treated roots. This result suggests that both the endodermis and exodermis may contribute to the regulation of water uptake into roots, but why Lp decreased more rapidly in PEG-treated root than in NaCl-roots is unknown. An increase in the activity of POD, which is involved in modifying cell wall components and detoxification of highly reactive molecules, is a common response to oxidative and abiotic stresses, but little is known about how POD participates in the formation of Casparian bands. Sancho et al. (1996) reported that total POD activity increased in salt-adapted cells and may be associated with the changed mechanical properties of the cell wall. A recent study suggested that a POD inhibitor blocked Casparian band formation and a specific POD, PER 64, was identified to colocalize with CASP1 protein in both early and mature Casparian band in Arabidopsis root (Halpin 2013; Lee et al. 2013). The present results showed that the total activity of POD in the cell wall of primary roots was elevated in both NaCl- and PEG-treated roots. Both salt and osmotic stresses are known to produce secondary oxidative stress. This increase of POD activity under stress condition could be related with both antioxidant role and cell wall modification. To further know the specific effect of POD activity in cell wall modification, we examined the apoplastic H2O2 in the cell wall of the endodermis and exodermis of roots grown under stress and control conditions by using a cerium chloride assay. Previous studies indicated that PODs might use localized superoxide-derived H2O2 for Casparian strip formation. Our data showed that NaCl and PEG treatments increased the H2O2-induced electron-dense precipitates of cerium perhydroxide at the Casparian strip position of endodermis. In the epidermis cell wall of PEGtreated roots, the electron-dense precipitates were apparently higher than those of NaCl-treated and control roots. Although the precise role of POD in cell wall modification of the Casparian band is unknown, it seems probable that this enzyme by use of localized superoxide-derived H2O2 would regulate production of phenolics, which are components of lignin and suberin in the cell wall. The extent to which plants respond to external parameters by increasing POD-regulated lignification or suberization in exodermal and endodermal cells remains to be elucidated. In conclusion, this study showed that in maize seedling roots, the locations of Casparian bands and suberin lamellae in the endodermis and exodermis were closer to the root tip under both salt and osmotic stress conditions. NaCl salinity, especially a high concentration of salt, impacted on the cell
walls of the outer cortical cells earlier than on the endodermal cells (Fig. S4), whereas PEG-induced osmotic stress caused cell wall suberization in both endodermal and exodermal cells almost simultaneously. In PEG-treated roots, Lp decreased more rapidly than in NaCl-treated roots. How plants developed these different strategies to modify the exodermal and endodermal cell walls in response to different abiotic stresses and whether this is a common mechanism among plants to adapt abiotic stresses remain to be studied. In the future, the stress responses of mutants and transgenic plants will be used to explore how plants regulate cell wall composition to form the exodermal and endodermal barriers in response to different environmental stresses. Studies of the activity of enzymes, such as POD and phenylalanine ammonia lyase, which are involved in modifying cell wall components, are in progress. Acknowledgments The authors are indebted to Mr. Xin Yu for the measurement of hydraulic conductivity and Mr. Xiao-Yan Gao for helping on microscope analysis. This paper was supported by the National Basic Research Program of China (2011CB710902), the China Manned Space Flight Technology Project, and the Strategic Pioneer Projects of CAS (XDA04020202). Conflict of interest The authors declare that they have no conflict of interest.
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ORIGINAL ARTICLE
Apoplastic barrier development and water transport in Zea mays seedling roots under salt and osmotic stresses Jie Shen & Guoxin Xu & Hui Qiong Zheng
Received: 12 September 2013 / Accepted: 16 June 2014 # Springer-Verlag Wien 2014
Abstract The development of apoplastic barriers was studied in Zea mays seedling roots grown in hydroculture solution supplemented with 0–200 mM NaCl or 20 % polyethylene glycol (PEG). Casparian bands in the endodermis of both NaCl- and PEG-treated roots were observed closer to the root tip in comparison with those of control roots, but the cell wall modifications in the endodermis and exodermis induced by salt and osmotic stresses differed. High salinity induced the formation of a multiseriate exodermis, which ranged from several cell layers to the entire cortex tissue but did not noticeably influence cell wall suberization in the endodermis. In contrast, osmotic stress accelerated suberization in both the endodermis and exodermis, but the exodermis induced by osmotic stress was limited to several cell layers in the outer cortex adjacent to the epidermis. The hydrostatic hydraulic conductivity (Lp) had decreased significantly after 1 day of PEG treatment, whereas in NaCl-treated roots, Lp decreased to a similar level after 5 days of treatment. Peroxidase activity in the roots increased significantly in response to NaCl and PEG treatments. These data indicate that salt stress and osmotic stress have different effects on the development of apoplastic barriers and water transport in Z. mays seedling roots.
Keywords Apoplastic barriers . Osmotic stress . Salt stress . Water transportation . Zea mays
Handling Editor: Néstor Carrillo Electronic supplementary material The online version of this article (doi:10.1007/s00709-014-0669-1) contains supplementary material, which is available to authorized users. J. Shen : G. Xu : H. Q. Zheng (*) Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, 200032 Shanghai, China e-mail: [email protected]
Introduction Roots of all vascular plants possess an endodermis, which occupies an interfacial position between ion absorbing and transporting tissue and provides a barrier to the apoplast of ions and other solutes (Bonnett 1967; Peterson et al. 1993; Naseer et al. 2012; Lee et al. 2013). According to Kroemer (1903) followed by Robards and Jackson (1975) and Geldner (2013), four developmental stages of the endodermis can be distinguished: (1) the pro-endodermis, (2) the primary stage endodermis with the Casparian band, (3) the secondary stage endodermis with the deposition of suberin lamellae on the internal wall surface, and (4) the tertiary stage endodermis with heavy U-shaped cell wall deposition on the inner tangential and the radial cell walls of the endodermal cells. Apoplastic transport in plant roots is blocked when they form Casparian strips (Schreiber et al. 1999; Ranathunge and Schreiber 2011; Halpin 2013). Experimental injuring of the endodermis indicates that the Casparian band has little effect on the movement of water, but it largely prevents the movement of ions, and the main resistance to water movement lies in the living tissues of the root (Peterson and Steudle 1993). In addition to an endodermis, many plants develop an exodermis, which is the outermost layer(s) of the cortex and undergoes cell wall modifications with the deposition of Casparian bands and suberin lamellae. Plant response to environmental stimuli by modifying the degree of suberization and lignification of root cell walls has been extensively studied (Negrel et al 1993; Bolwell et al. 1997; Franke and Schreiber 2007; Bernards and Lewis 1998; Geldner 2013; Halpin 2013). Apoplastic barriers, which are composed of suberin and restrict water and nutrient loss and prevent the invasion of pathogens, were strengthened by various stress conditions (Höfer et al. 2008; Krishnamurthy et al. 2009). The amounts of endodermal suberin and lignin increase 1.5-fold and 2.0-fold, respectively, in corn roots
S. Jie et al.
treated with NaCl and polyethylene glycol (PEG) (Schreiber et al. 1999). Thomas et al. (2007) reported strong negative correlations between the amount of the aliphatic component of root suberin and plant mortality induced by Phytophthora sojae. These modifications of cell walls lead to a decrease in radial water transport in the roots (reviewed by Hose et al. 2001; Schreiber et al. 2005; Franke and Schreiber 2007), such as the exodermis, which increases resistance to radial water movement, making it more difficult for the root to take up water (Peterson and Enstone 1996). The physiological importance of apoplastic barriers in many plants is well known (Zimmermann et al. 2000; Schreiber et al. 2005; Naseer et al. 2012; Lee et al. 2013). However, how environmental factors influence the formation of apoplastic barriers remains enigmatic. Deeper insight into the development of apoplastic barriers is required to elucidate the factors that determine barrier properties. In the present research, we studied exodermal and endodermal development in relation to the formation of apoplastic barriers in growing Zea mays roots under salt stress and osmotic stress conditions, respectively. Our data indicated that the properties of apoplastic barriers in the root are tissue specific and regulated in response to different environmental stress conditions.
Materials and methods Plant material Seeds of maize (Z. mays L. cv. Suyu NO.1) were surface sterilized with a solution of sodium hypochlorite (0.5 %, w/ v) and Tween 20 (0.75 %, w/v) for 10 min. After thorough rinsing with distilled water, the seeds were imbibed and germinated on filter paper moistened with distilled water for 2 days at 26–27 °C in the dark. Seedlings with roots about 3 cm long were chosen and grown in culture chambers containing 5-L Hoagland nutrient hydroculture solution (Hoagland and Arnon 1938). The control consisted of basic Hoagland solution. Salt and drought treatments comprised the basic nutrient solution supplemented with 100–200 mM NaCl and 20 % PEG 6000 (w/v), respectively. The total volume of the solution in each culture chamber was kept constant by adding double-distilled water to compensate for water loss through plant evapotranspiration. The osmotic potential of the culture solutions was measured with a vapor pressure osmometer (Wescor, Logan, UT, USA). The seedlings were grown in a greenhouse (26–27 °C) with a 16-h photoperiod at 200 μmol m−2 s−1 photon flux rate and harvested 8–12 days after initiation of treatment. Histochemical staining for light microscopy The development of root endodermal and exodermal cells was examined with a series of freehand sections. Casparian bands
were detected by staining with the fluorescent dye neutral red and toluidine blue, or berberine hemisulfate, as described by Brundrett et al. (1988), and suberin lamellae were detected by staining with Sudan III, as described by Schreiber et al (1999). Measurement of hydraulic conductivity Root hydrostatic hydraulic conductivity (Lp) was measured in accordance with the method of Miyamoto et al. (2001). Briefly, roots were enclosed in a steel chamber, and pneumatic pressure was applied to the root bath medium (water). The pressure in the chamber (Pgas) was raised in steps of 0.05 up to 0.3 MPa above atmospheric pressure. The exuded sap was collected, and the volume of the exuded sap (Jv; in m3 m−2 s−1) was calculated. The value of Lp was calculated according to the formula: . Lp ¼ J v Pgas þ σ•ðΠ b −Π s Þ
where σ is the root reflection coefficient for nutrient salts in the xylem, which was estimated to be σ=0.4, and Πb and Πs were the osmotic pressure of the bath medium and sap exuded from individual plants, respectively. At a high value of Pgas (≥0.1 MPa), the effect of the osmotic term is not pronounced, so σ, Πb, and Πs can be ignored (Miyamoto et al. 2001). Lp was determined from plots of Jv against Pgas. Histochemical localization of peroxidase activity Peroxidase (POD) activity was monitored as described by Ros-Barcelo et al. (2002) with modifications. Root sections were incubated for 20 min at 25 °C in a staining solution composed of 0.1 mg mL−1 tetramethylbenzidine (TMB)-HCl in 50 mM Tris-acetate buffer (pH 5.0). Staining was monitored in 25 sections selected randomly from 20 to 30 roots.
Results Growth and development of primary roots under salt and osmotic stresses Both salinity and drought stresses result in decreased availability of water to plant cells and affect plant growth and development. To understand how Z. mays plants changed their growth and development to counteract the decreased availability of water under these stresses, we treated seedlings with low water potential (ψw) by using nutrient solutions supplemented with serial concentrations of NaCl or 20 % PEG 6000. After culture in nutrient solution containing 50, 100, 150, or 200 mM NaCl for 8–12 days, the treated seedlings exhibited
Apoplastic barrier development and water transport in Zea mays
obvious stress symptoms but survived up to 200-mM NaCl treatment (Fig. S1). The total root length and the sites of the first lateral root and root hair development were significantly affected by NaCl at concentrations exceeding 100 mM (Fig. 1, Table S1). The total length of primary roots under the NaCl- or PEG-treated conditions relative to that under the control condition was significantly reduced by 47 and 62 %, respectively (Table 1). The length of the elongation region of 100 mM NaCl- or 20 % PEG-treated roots was reduced by 80 and 88 %, respectively, whereas the size of root caps showed no significant difference. The distance from the site of the first lateral root to the root apex under the NaCl- or PEG-treated conditions was reduced by 83 and 51 %, respectively. The distance from the site of the first lateral root to the root apex under NaCl stress was about 3-fold shorter than that under PEG stress. In contrast, the distance from the site of root hair development to the root apex in PEG-treated roots was 1.7-fold lower than that in NaCl-treated roots (Table 1). These results indicate that young maize seedlings respond differentially to salt and drought stress. Hydraulic conductivity of seedling roots under salt and osmotic stresses The Lp value was not significantly affected by 100-mM NaCl treatment after 1 day but was reduced by 34.5 % after treatment for 5 days compared with that in the control roots. The Lp values in 20 % PEG-treated roots decreased by 69 and 93 % after 1 and 5 days of treatment, respectively, compared with those in the control roots (Fig. 2). These data showed that PEG treatment decreased Lp within a short period (less than 1 day). Treatment with NaCl also affected the Lp value in the roots but only after a longer period in comparison with PEG treatment. Development of endodermis and exodermis under salt and osmotic stresses Previous studies indicate that the apoplasmic water permeability might depend on the developmental state of the root (Peterson et al. 1993). To evaluate how the hydraulic resistance of the root changes in response to external stresses (e.g., drought and salinity), the formation of apoplastic barriers (Casparian bands and suberin lamellae) in the endodermis and exodermis under osmotic and salinity stresses in young roots was examined. The roots of seedlings grown under NaCl and PEG treatments and the control conditions were fixed, and cross sections about 0.1-mm thick were cut from the root tips continuously. Each section was examined with an epifluorescence microscope to observe the cell wall modifications (Fig. S2). When the cells of an 8-day-old seedling grown under the control condition were arranged in sequence basipetally from the root tip, the endodermal cells were observed to
Fig. 1 Phenotype of Zea mays seedlings (8 days old) cultured under the control condition, 100 mM NaCl condition, and 20 % PEG condition, respectively. Bar 5 cm
be at the primary stage with the Casparian strip present in the region about 13–38 mm from the root tip (Fig. 3a). Endodermal cells at the secondary stage, showing deposition of suberin lamellae on the internal wall surface, were observed in the region about 38–70 mm from the root tip (Figs. 3b and 4). About 95–110 mm from the root apex, the endodermal cells displayed a strong U-shaped cell wall deposition on the inner tangential and the radial cell walls and had developed into the tertiary stage (Fig. 3c). The sites of endodermal Casparian bands were observed closer to the root tip in both NaCl- and PEG-treated roots in comparison with those under the control condition. The distance from the root tip to the site of the first lateral root in control roots was about 70 mm but was decreased to about 11 and 34 mm in 100-mM NaCl- and 20 % PEG-treated roots, respectively. The Casparian strip had developed into the tertiary stage at about 37 mm from the apex of PEG-treated roots compared with about 95 mm in control roots and about 85 mm in the NaCl-treated roots (Fig. 4). Suberin lamellae, corresponding to the Casparian strip, were detected in the endodermal cell walls of salt- and osmoticstressed roots and control roots by staining with Sudan III, a lipophilic dye commonly used to visualize suberin deposition. Compared with those in control roots (Fig. 5a), the Casparian strip in NaCl-treated roots showed decreased staining, whereas PEG-treated roots exhibited a distinct increase in staining intensity (Fig. 5b, c). This result suggests that suberin deposition in the cell wall during development of endodermal cells might be differentially modified by salt and osmotic stresses. The cross sections with secondary stage endodermis were selected for staining with the berberine–aniline blue procedure, which is a rapid and precise method for visualizing lignified cell walls, Casparian bands, suberin lamellae, and callose (Brundrett et al. 1988). When the sections from control
S. Jie et al. Table 1 Different effect of salt and osmotic stress on the development of Zea mays seedling roots (mean±SD, n=200) Treatment
Total lengtha (cm)
Root tip lengthb (cm)
Root cap size (μm)
First lateral rootc (cm)
Control 100 mM NaCl 20 % PEG
21.68±5.1 11.52±1.36 8.2±1.19
1.28±0.32 0.26±0.16 0.15±0.02
418±96 398±28 335±30
6.85±2.68 1.14±0.67 3.35±0.83
a
Total length of root
b
Distance from the root apex to the site of root hair appearance
c
Distance from the root apex to the site of the first lateral root appearance
roots were examined under ultraviolet light, tracheary element walls fluoresced intense yellow-white, the endodermal Casparian band fluoresced yellow-green, and cortical cells fluoresced blue (Fig. 6a). Staining of the endodermal Casparian bands in NaCl-treated roots was not noticeably different from those in control roots (Fig. 6a, b), whereas almost a full ring of endodermal cells with suberin lamellae was observed in PEG-treated roots (Fig. 6c). Intense yellowish green fluorescence was observed in all cell walls of parenchyma cells in the central and outer cortex of NaCl-treated roots (Figs. 6b and S3). A high concentration of NaCl promoted formation of exodermis in the cortex from several cell layers to the whole tissue (Figs. S3 and S4). This multiseriate exodermis also formed in PEG-treated roots but was limited to three to four layers of the outer cortical cells and never extended to the whole tissue (Fig. 6c). Peroxidase activity of seedling roots under salt and osmotic stresses Histochemical localization of POD by staining with the TMB reagent showed that POD activity in the primary root was strongly affected by treatment with 100 mM NaCl and 20 %
PEG. The activity of POD in the cell walls of the endodermis and vascular cylinder of both NaCl- and PEG-treated roots was visibly increased (Fig. 7b, c) in comparison with that of the control condition (Fig. 7a). In contrast, the activity of POD in the cell wall of epidermal cells increased under PEG treatment (Fig. 7c) but showed no obvious change under NaCl treatment (Fig. 7b) in comparison with that under the control condition (Fig. 7a). A recent study indicated that PODs used localized superoxide-derived H2O2 for Casparian strip formation (Lee et al. 2013). To test whether the production of apoplastic H2O2 in the endodermis is affected by NaCl and PEG treatment, we used the cerium chloride assay, in which cerium chloride forms electron-dense precipitates in the presence of H2O2 through formation of cerium perhydroxide. With this assay, we observed apparently localized cerium precipitations at the position of Casparian strips of endodermal cells in NaCl- and PEGtreated roots in comparison with that of control roots (Figs. S5 and S6). Cerium precipitations were also observed at the position of inner cell wall of epidermal cells in PEG-treated roots (Fig. S7f, i) but not in the NaCl-treated and the control roots (Fig. S7d, e, g, h). These results indicated that distribution of apoplastic H2O2 in the endodermis and exodermis was also differentially affected by NaCl and PEG treatment.
Discussion
Fig. 2 Effects of salt and osmotic stress on the hydraulic conductivity (Lp) of Zea mays seedling roots. Con control, NaCl 100-mM NaCl treatment, PEG 20 % PEG treatment
Several previous studies suggest that rapid responses (within a few hours of application) to salt often resemble responses to low ψw imposed using nonionic solutes (e.g., PEG 6000), whereas longer-term responses (from days to weeks) are more salt specific (Carillo et al. 2011; Munns 2002, 2011; Verslues et al. 2006). At the cellular level, the difference in tissue responses to isotonic salinity and osmotic treatments is also observed at the minute timescales, both in root (Chen et al. 2005) and shoot (Shabala 2000) tissues. The most pronounced structural responses of plants to salinity and osmotic stresses are often related to cell wall modification. Some fundamental
Apoplastic barrier development and water transport in Zea mays
Fig. 3 The Casparian strip in a Zea mays root stained with neutral and toluidine blue and viewed under UV light with an epifluorescence microscope. a, b, c Florescence micrographs showing endodermal
Casparian bands. d Diagram of a Zea mays seedlings, and the marked regions are correspondingly shown in a, b, c. X xylem, En endodermal cells, Co cortex cells. Bars 50 μm in a, b, and c
information regarding cell wall modification under stress conditions has been known for a long time. For example, salinity accelerates development of endodermis and induces formation of uniseriate, biseriate, or multiseriate exodermis in roots of many plant seedlings including monocots and dicots and members of primitive and advanced plant families (Reinhardt and Rost 1995; Degenhardt and Gimmler 2000; Karahara et al. 2004). However, it is only recently that significant new insights regarding the underlying molecular factors for localized lignin deposition in the endodermis have come to light (Lee et al. 2013). We are still largely ignorant about how plants use this mechanism of localized cell wall modification to cope with different stresses. Our results showed that the cell wall modifications in the developing endodermis of Z. mays seedling roots were affected by both NaCl and PEG treatment, but the effects of salt (NaCl) and osmotic (PEG) stress on the cell wall modifications of endodermal and exodermal cells differed markedly. Initiation of the Casparian strip was shown to be correlated with development of the phloem and xylem
(Wilcox 1954; Martinka et al. 2012), where root hairs develop from epidermal cells. The investigation by Haas and Carothers (1975) suggests that in Z. mays, there is a single precisely determined site at which Casparian strip formation occurs and that there is no prior or subsequent shift in this position. Our present results showed that the site of root hair development was closer to the root apex in PEG-treated roots than that in NaCl-treated roots. Comparisons of the development of endodermal cells in NaCl- and PEG-treated roots indicated that suberization in the endodermis was more strongly promoted by osmotic stress than that by salt stress (Fig. 6). Exposure to salinity induced apparent suberization of the cell wall from one cell layer to the entire cortex tissue with increasing concentration of NaCl, whereas osmotic stress induced formation of an exodermis limited to three to four layers of the outermost cortical cells. These results suggest that roots might adopt different mechanisms for the development of the Casparian strip and the deposition of suberin lamellae in the exodermis and endodermis to counteract salt and drought stresses. NaCl salinity induced suberization of the exodermis before suberization of the Casparian strip in the endodermis, whereas PEGinduced osmotic stress caused intensified suberization of exodermal and endodermal cell walls almost simultaneously. Salinity and drought cause intensified suberization in exodermal cell walls of several plant species (e.g., cotton, citrus, and sorghum) (Walker et al. 1984; Cruz et al. 1992; Reinhardt and Rost 1995; Simone et al. 2003), but roots that develop different strategies to cope with different stresses have not been reported. In the present study, no exodermis was observed at 95 mm from the root apex in control roots. However, in salt (100 mM NaCl)-treated roots, the exodermis was observed as close as 30–40 mm from the root apex, which coincides with the region that root hairs and lateral roots begin to develop. These sites will be critical for uptake of mineral nutrients and water by the root. When roots were treated with 200 mM NaCl, suberization of the exodermis was observed almost from the beginning of the region of root hair development. We hypothesize that the presence of a multilayer
Fig. 4 Distance from the root apex to the point of the endodermal Casparian strip appearance (stage A) and the endodermal suberin lamellae appearance in secondary stage (stage B) and in tertiary stage (stage C). Con control, NaCl 100 mM NaCl, PEG 20 % PEG
S. Jie et al. Fig. 5 Cross sections were made at the site of the first lateral root appearance and stained with Sudan III. The roots were grown under control (a), 100 mM NaCl (b), and 20 % PEG (c) conditions, respectively. En endodermal cells, X xylem. Bars 50 μm in a, b, and c
Fig. 6 Cross sections were made at the site of the first lateral root appearance and stained with berberine hemisulfate and aniline blue to illustrate the effects of salt and osmotic stresses on exodermal and endodermal development. a Control. b 100mM NaCl treatment. c 20 % PEG treatment. Co cortex, X xylem, En endodermal cells, Ep epidermal cells. Bars 100 μm in a, b, and c
exodermis in the outer cortex protects the inner cortex and vascular cells from exposure to the high ion concentration in the external solution; thus, suberization of the endodermis was not significantly accelerated in NaCl-treated roots compared with that in PEG-treated roots. The process of water movement through the root and the location of the major barrier(s) to water movement are still subjects of debate. The Casparian band, of which components are hydrophobic, has been assumed to be a barrier of radial Fig. 7 Histochemical localization of peroxidase in cross sections at the region of the first lateral root appearance, stained with the TMB reagent. a Control. b 100 mM NaCl. c 20 % PEG. Co cortex, X xylem, En endodermal cells, Ep epidermal cells. Bars 50 μm in a, b, and c
movement of water in the root (Frensch et al. 1996). However, Peterson et al. (1993) argued that the endodermis (with Casparian bands only) was not a major barrier to water movement into the root by determining the changes in hydraulic conductivity after injuring the endodermis (Peterson et al. 1993; reviewed by Steudle and Peterson 1998). One consistent observation regarding the radial cell-to-cell flow of water in the root is that suberin lamellae are barriers to water movement. Formation of an exodermis with suberin lamellae may
Apoplastic barrier development and water transport in Zea mays
add to the overall resistance to water flow. An exodermis is usually absent in young maize roots grown in hydroponics (Steudle and Peterson 1998; Zimmermann and Steudle 1998) and is induced by NaCl and PEG stress treatments. In the present study, the hydraulic conductivity (Lp) in both NaCland PEG-treated roots was significantly decreased, but Lp decreased more rapidly in response to PEG treatment than was observed in NaCl-treated roots. This result suggests that both the endodermis and exodermis may contribute to the regulation of water uptake into roots, but why Lp decreased more rapidly in PEG-treated root than in NaCl-roots is unknown. An increase in the activity of POD, which is involved in modifying cell wall components and detoxification of highly reactive molecules, is a common response to oxidative and abiotic stresses, but little is known about how POD participates in the formation of Casparian bands. Sancho et al. (1996) reported that total POD activity increased in salt-adapted cells and may be associated with the changed mechanical properties of the cell wall. A recent study suggested that a POD inhibitor blocked Casparian band formation and a specific POD, PER 64, was identified to colocalize with CASP1 protein in both early and mature Casparian band in Arabidopsis root (Halpin 2013; Lee et al. 2013). The present results showed that the total activity of POD in the cell wall of primary roots was elevated in both NaCl- and PEG-treated roots. Both salt and osmotic stresses are known to produce secondary oxidative stress. This increase of POD activity under stress condition could be related with both antioxidant role and cell wall modification. To further know the specific effect of POD activity in cell wall modification, we examined the apoplastic H2O2 in the cell wall of the endodermis and exodermis of roots grown under stress and control conditions by using a cerium chloride assay. Previous studies indicated that PODs might use localized superoxide-derived H2O2 for Casparian strip formation. Our data showed that NaCl and PEG treatments increased the H2O2-induced electron-dense precipitates of cerium perhydroxide at the Casparian strip position of endodermis. In the epidermis cell wall of PEGtreated roots, the electron-dense precipitates were apparently higher than those of NaCl-treated and control roots. Although the precise role of POD in cell wall modification of the Casparian band is unknown, it seems probable that this enzyme by use of localized superoxide-derived H2O2 would regulate production of phenolics, which are components of lignin and suberin in the cell wall. The extent to which plants respond to external parameters by increasing POD-regulated lignification or suberization in exodermal and endodermal cells remains to be elucidated. In conclusion, this study showed that in maize seedling roots, the locations of Casparian bands and suberin lamellae in the endodermis and exodermis were closer to the root tip under both salt and osmotic stress conditions. NaCl salinity, especially a high concentration of salt, impacted on the cell
walls of the outer cortical cells earlier than on the endodermal cells (Fig. S4), whereas PEG-induced osmotic stress caused cell wall suberization in both endodermal and exodermal cells almost simultaneously. In PEG-treated roots, Lp decreased more rapidly than in NaCl-treated roots. How plants developed these different strategies to modify the exodermal and endodermal cell walls in response to different abiotic stresses and whether this is a common mechanism among plants to adapt abiotic stresses remain to be studied. In the future, the stress responses of mutants and transgenic plants will be used to explore how plants regulate cell wall composition to form the exodermal and endodermal barriers in response to different environmental stresses. Studies of the activity of enzymes, such as POD and phenylalanine ammonia lyase, which are involved in modifying cell wall components, are in progress. Acknowledgments The authors are indebted to Mr. Xin Yu for the measurement of hydraulic conductivity and Mr. Xiao-Yan Gao for helping on microscope analysis. This paper was supported by the National Basic Research Program of China (2011CB710902), the China Manned Space Flight Technology Project, and the Strategic Pioneer Projects of CAS (XDA04020202). Conflict of interest The authors declare that they have no conflict of interest.
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