听lignification_suberization

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Lignified/suberized root exodermis contributes to metal tolerance 647

allow sufficient oxygen to reach submerged roots (Youssef Table 1. Characteristics of the six mangrove species used in the and Saenger 1996, Pi et al. 2009). Extensive aerenchyma in presentstudy.

roots provides a low-resistance internal pathway for O2 trans-SpeciesFamilyTidal position in Leizhou, port within roots (Armstrong and Beckett 1987, Kludze et al. Guangdong, China1993, Jackson and Armstrong 1999). In addition, a barrier to A. ilicifoliusAcanthaceaeUpper or middle reaches radial oxygen loss in basal root zones diminishes oxygen leak-of estuarine rivers, pioneer age during long-distance transport of oxygen within roots and species

enhances oxygen diffusion towards root tips (Colmer 2003, A. corniculatumMyrsinaceaeSeaward regions, pioneer Colmer and Pedersen 2008). It is generally accepted that the species

performance of this impermeable barrier is mainly related to A. marinaVerbenaceaeSeaward, mangrove fringe,

pioneer species

the structure of the exodermis, as well as to quantitative varia-B. gymnorrhizaRhizophoraceaeBack mangroves, landward tions in lignin/suberin distribution within the exodermal cell or mid-intertidal

walls (Soukup et al. 2007).K. obovataRhizophoraceaeMiddle but also extends Moreover, this impermeable root barrier regulates the into the transitional

foreshore

fluxes of gases, water and solutes at the soil–root interface R. stylosaRhizophoraceaeMid-intertidal

(Garthwaite et al. 2006, Meyer et al. 2011), and also plays

important roles in protecting plants from biotic and abiotic

stresses (Degenhardt and Gimmler 2000, Pollard et al. 2008). province, China (21°31′N and 109°41′E) (Table 1). After ger-Generally, lignin/suberin deposition within the exodermis sig-mination, the seedlings were kept in a greenhouse at a tem-nificantly contributes to the formation of an apoplastic trans-perature of 25 ± 5 °C and a light intensity of 480 µmol m?2 s?1, port barrier. It has been reported that salt-tolerant rice cultivars and frequently flooded (12 h flooded and drained for another often exhibit more extensive lignification/ suberization than 12 h to simulate a 12/12 h high/low tidal cycle) with brackish salt-sensitive cultivars (Krishnamurthy et al. 2009). Our recent water (salinity 10‰, w/v, prepared by the addition of 10 g sea results (Cheng et al. 2010, 2012) also reported that mangroves salt (Sigma-Aldrich, Shanghai, China) per liter of water). The (e.g., Bruguiera gymnorrhiza (L.) Poir and Rhizophora stylosa seedlings were allowed to grow until the following March. Then, Griff) appear to increase lignification under metal- contaminated uniform overwintered seedlings of the six mangroves were conditions, indicating a defense response to metal toxicity. selected for the measurements of root anatomy and metal tol-Previous studies (Pi et al. 2009) illustrated the differences erance. The seedlings of each species were uniform in size: in root anatomy (e.g., exodermal structure, lignification/ A. marina and A. corniculatum were ~7–8 cm in stem height and suberization) among different mangrove species using micros-7–9 cm in root length; A. ilicifolius was ~10 cm in height and copy. However, the measurements of lignin/suberin content and 8–9 cm in root length; B. gymnorrhiza, K. obovata and R. stylosa composition within the roots of mangroves are still lacking. It were ~10–14 cm in stem height and 9–10 cm in root length.would obviously be of great interest to evaluate metal uptake

and tolerance in mangrove plants from the aspect of root anat-Comparisons of root anatomy and lignification/

omy and lignification/suberization.suberization among six mangrove species

The present study, therefore, aims to (i) compare metal Uniform overwintered seedlings of the six mangrove spe-uptake and tolerance in six common mangrove species domi-cies as mentioned above were selected for the measurement nant along the South China coast, (ii) evaluate the relations of root anatomical features. Lateral roots with similar size between metal tolerance and root anatomy and lignification/(~6–7 cm in length and with similar diameter ~0.8–1.2 mm suberization, and (iii) illustrate the effects of the lignified/in basal root regions) were selected for anatomical micro-suberized exodermis on metal uptake, translocation and toler-analysis, and free-hand cross-sections were made at differ-ance in mangrove plants.ent distances from the root tip (1, 3 and 5 cm). Firstly, the

sections were examined under scanning electron micros-

Materials and methodscopy and photographed (Deng et al. 2009). Then, a pho-Preparation of plant materialstomicroscope equipped with fluorescence attachments was

employed to detect lignification and suberization (Zeier

In the year prior to this study (2011), propagules or seeds of six et al. 1999, Armstrong and Armstrong 2005, Thomas et al. mangrove plants (three foreshore pioneer mangroves and three 2007). Sections were stained with phloroglucinol and con-rhizophoraceous mangroves), namely Aegiceras corniculatum centrated hydrochloric acid to show lignification (viewed with (Linn.) Blanco, Acanthus ilicifolius L., Avicennia marina (Forsk.) white light). For suberization observation, the sections were Viern., B. gymnorrhiza, Kandelia obovata Sheue, Liu & Yong and unstained, or stained with toluidine blue and neutral red, then R. stylosa, were collected from Leizhou byland, Guangdong viewed with blue light; lipid suberin within the exodermis was

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648 Cheng et al.

detected using fluoral yellow 088 (FY 088) (viewed with UV prepared soil) included three groups: low (Pb 50, Zn 100, Cu light) (Brundrett et al. 1991); the existence of suberin poly-50), medium (Pb 100, Zn 200, Cu 100) and high (Pb 200, mer was also examined using the method of acid digestion Zn 400, Cu 200). The concentrations selected for use in the (placed in concentrated sulfuric acid and incubated for 48 h) pot trial were based on a previous study (Liu et al. 2009), as described by Thomas et al. (2007).which presented evidence of metal inhibition distinct enough Lignin and suberin within the exodermal cell walls of lateral to distinguish between species and treatments. Another set of root segments from six mangrove species were examined pots without metals was also prepared as the control group. by gas chromatography and mass spectrometry (GC–MS). For each species, four pots (two seedlings per pot) per treat-The details of the methods of lignin/suberin analysis have ment were used, and one pot (average data of living seedlings been described by Schreiber et al. (1994). Root segments in each pot) was considered as one replication. The seedlings (0–5 cm, with similar diameters of 0.8–1.2 mm) were incu-were frequently flooded (12/12 h high/low tidal cycle) with bated in a citric buffer containing cellulase and pectinase. brackish water (salinity 10‰, prepared with the addition of After 2 weeks of enzymatic digestion, exodermal cell walls 10 g of sea salt per liter of water), and fresh water was added were separated. Six samples per species out of 30 pooled twice a week to compensate for evaporation.

seedlings were analyzed, half of them (n = 3, and each sam-After 120 days of exposure to metals, the seedlings were ple was collected from five seedlings) were prepared for lig-harvested and washed with deionized water. Growth param-nin examination, and the remaining samples were used for eters, such as survival rate, height, number of fully expanded suberin analysis. The thioacidolysis procedure was used for leaves and biomass yield, were taken immediately after har-the detection of lignin according to Lapierre et al. (1991). Cell vest. The seedlings were then dried for 1 week at 60 °C, to wall isolates were stirred in a mixture of BF3 etherate, ethane-a constant weight, for the calculation of the total biomass thiol and dioxane at 100 °C for 4 h in an argon atmosphere, (which is a summation of root, stem and leaf, but without then the mixture was cooled, diluted with 2 ml of water and propagules). The index of metal tolerance was calculated by extracted three times with 3 ml of CHCl3 containing 20 µg comparing the rates of biomass production in soils with and of dotriacontane as the internal standard. Suberin was ana-without metal addition. The oven-dried samples (root and leaf, lyzed after the procedure of transesterification according to 0.2 g) were digested with a mixture of concentrated nitric Kolattukudy and Agrawal (1974). The isolated cell wall was acid and hydrogen peroxide (MacFarlane and Burchett 2002). added to 10% BF3 methanol solution and heated at 60 °C for The concentrations of metals in the digests were determined 24 h. After cooling, the solution was collected. The remain-using inductively coupled plasma–optical emission spectros-ing cell walls were washed three times with CHCl3 containing copy (ICP–OES). Blanks and standard materials (GBW-07063, 20 µg of dotriacontane as the internal standard. The solutions G sv-2, National Standard Materials Research Center, Beijing, were then combined and washed with 2 ml of saturated NaCl China) were tested for quality assurance.

solution, and the organic phase was separated and dried with

Na2SO4. Gas chromatographic analysis and mass spectromet-Metal uptake by excised roots with different root ric identification of the extracts of lignin and suberin were anatomy and lignification/suberization

performed as previously described in detail by Zeier and In order to investigate in more detail the function of root Schreiber (1997). The samples were converted to trimethyl-anatomy and lignification/suberization in metal uptake and silyl derivatives using N,N-bis-trimethylsilyltrifluoroacetamide tolerance, metal uptake by the excised roots was also deter-(BSTFA). Quantitative and qualitative sample analysis was mined (Hu et al. 2014). The experiment followed the previ-carried out by a gas chromatograph equipped with a flame ous study reported by Otte et al. (1989) with modification. ionization detector or coupled with a quadrupole mass selec-Root segments (apical 0–5 cm, diameter 0.8–1.2 mm) from tive detector, respectively.six mangroves were incubated in 5 mM MES buffer (pH = 5.5, Evaluation of metal uptake and tolerance by mangrovescontaining 0.5 mM Ca(NO3)2 and 19 µM Pb, 0.06 mM Zn and

0.06 mM Cu for 1 h). For each species, three samples pooled

A pot trial with addition of metals was employed to evaluate from 15 seedlings (n = 3, and each sample was collected metal uptake and tolerance by mangroves. Uniform overwin-from 5 seedlings) were analyzed. The metal concentration tered seedlings of the six mangroves described above were selected was based on our preliminary experiment, which is transplanted into plastic pots (20 cm diameter and 25 cm high enough to distinguish the difference in metal uptake by height) with 4 kg of prepared soil (50% silty clay loam, 40% the excised roots with different root anatomy. Subsequently, clean river sand, 10% organic peat moss) per pot. Metal treat-the segments were rinsed thoroughly with deionized water to ments were prepared by the addition of appropriate amounts remove absorbed metals from the root free space. Then, the of chloride: PbCl2, ZnCl2 and CuCl2. The concentrations concentrations of Pb, Zn and Cu in roots were determined by of heavy metals (milligrams heavy metals per kilogram dry ICP–OES as described above.

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Lignified/suberized root exodermis contributes to metal tolerance 649

Statistical analysisA thin exodermis with about one to three layers of loosely

packed cells was found in the apical regions of roots of A. marina,

Data on plant performances were tested for their normality and A. corniculatum and A. ilicifolius. The apical regions of roots of homogeneous variance prior to a parametric one-way analysis the three rhizophoraceous species also exhibited a thin exodermis of variance. If the differences were significant at the 5% proba-with low lignification/suberization. However, in the medium and bility level, the least significant difference (LSD) was calculated sub-basal regions of roots, significantly more lignified/suberized as the post hoc test to determine where the differences lay. cell walls within five to nine cell layers of the exodermis were The statistical analyses were performed using SPSS statistical observed in the rhizophoraceous species when compared with packages, and the figures were created using the PC-based the pioneer mangroves.

Origin program.The results from GC–MS also showed that lignin and

suberin deposition within the exodermis varied significantly

Resultsamong the six mangrove species studied (Figure 3). The Comparisons of root anatomy and lignification/dominant lignin degradation products were trithioethylated suberization within the exodermis among six mangrove aromatic monomers, corresponding to three typical lignin speciesunits: p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S). The

amounts of total lignin of the three rhizophoraceous species

Root anatomy and the structure of the exodermis of the man-ranged from 70 to 110 µg mg?1. Compared with the three groves studied are presented in Table 2 and Figures 1 and 2. rhizophoraceous species, significantly lower levels of lignin Table 2. Characteristics of outer cell layers (exodermis) in basal, medium and apical roots of the six mangroves (presence of lignification/suberization: –, not found; +, thin; ++ , thick; +++ , very thick).Mangrove speciesPresence of lignified/suberized wallsNo. of outer cell layers

Apical rootMedium rootBasal rootApical rootMedium rootBasal root

A. ilicifolius–++1–21–31–3

A. corniculatum–++2–32–32–3

A. marina–++1–32–32–3

B. gymnorrhiza+++++++2–35–65–8K. obovata–++ ++2–34–65–8R. stylosa+++++++1–35–7

6–9

Figure 1. Cross-sectional scanning electron micrographs of the roots (5 cm from the root tip) in six mangrove plants, comprising (a–c) three pioneer species (A. ilicifolius, A. corniculatum and A. marina) and (d–f) three rhizophoraceous species (B. gymnorrhiza, K. obovata and R. stylosa).

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Cheng et al.

Figure 2. Bright-field and fluorescence microscope pictures of the root cross-sections of two selected mangroves, pioneer species A. corniculatum and rhizophoraceous species R. stylosa, bar = 100 µm. (a–f) Stained with phloroglucinol and hydrochloric acid to show lignification, black arrows. (a–c) Apical, medium and basal roots (1, 3 and 5 cm from the root tip, respectively) of A. corniculatum with low lignification; (b–f) R. stylosa, 1, 3 and 5 cm from the root tip, with a thick lignified exodermis in the medium and basal regions of roots. (g–n) The differences in suberization within basal roots between A. corniculatum (g, i, k and m) and R. stylosa (h, j, l and n), white arrows. (g and h) Unstained, viewed with blue light; (i and j) resisting digestion with concentrated sulfuric acid indicating suberin polymers. (k and l) Stained with toluidine blue and neutral red and viewed with blue light; (m and n) stained with FY 088 and viewed with UV light.

were detected in A. marina, A. corniculatum and A. ilicifolius alcohols and 2-hydroxyacids. ω-Hydroxyacids (40–53%) (15–52 µg mg?1) (Figure 3a). For all species investigated, and diacids (24–33%) were dominant substance classes in G and S lignin were prominent components, whereas H lig-all species, except for A. ilicifolius. Carboxylic acids, followed nin was undetectable or found in very low concentrations by ω-hydroxyacids and 2-hydroxyacids, were the dominant (Figure 3b). As for aliphatic suberin, the highest suberin compounds in A. ilicifolius (Figure 3d).

was observed in B. gymnorrhiza (143 µg mg?1), followed

by K. obovata (127 µg mg?1), R. stylosa (106 µg mg?1), Differences in metal uptake and tolerance

A. marina (63 µg mg?1), A. corniculatum (54 µg mg?1) and among mangroves

A. ilicifolius (19 µg mg?1) (Figure 3c). The aliphatic suberin After 120 days of exposure to mixed Pb, Zn and Cu, most within the exodermis of the mangroves consists of long-growth indices, including seedling height, leaf number and bio-chain (C16–C30) ω-hydroxyacids, diacids, carboxylic acids, mass, were directly inhibited by metals. However, the sensitivity Tree Physiology Volume 34, 2014Downloaded from http://wendang.chazidian.com/ at Zhejiang University on February 8, 2015