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Galactoglucomannan oligosaccharides alleviate cadmium stress in Arabidopsis
Journal of Plant Physiology 171 (2014) 518–524
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Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Physiology
Galactoglucomannan oligosaccharides alleviate cadmium stress in Arabidopsis Danica Kuˇcerová a,b , Karin Kollárová a,∗ , Ivan Zelko a , Zuzana Vatehová a , Desana Liˇsková a a b
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovakia Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, 845 23 Bratislava, Slovakia
a r t i c l e
i n f o
Article history: Received 8 July 2013 Received in revised form 26 November 2013 Accepted 10 December 2013 Available online 13 March 2014 Keywords: Arabidopsis thaliana Cadmium distribution in tissues Galactoglucomannan oligosaccharides Suberin lamellae development
s u m m a r y Our study focused on the mediatory role of galactoglucomannan oligosaccharides (GGMOs) in plant protection against cadmium stress, examined mainly on the primary root growth of Arabidopsis thaliana. The application of GGMOs diminished the negative effect of cadmium on root length, root growth dynamics and also on photosynthetic pigment content. We tested the hypothesis that the effect of GGMOs is associated with decreased cadmium accumulation or its modified distribution. Cadmium distribution was observed chronologically from the first day of plant culture and depended on the duration of cadmium treatment. First, cadmium was stored in the root and hypocotyl and later transported by xylem to the leaves and stored there in trichomes. The protective effect of GGMOs was not based on modified cadmium distribution or its decreased accumulation. In cadmium and GGMOs + cadmiumtreated plants, the formation of suberin lamellae was shifted closer to the root apex compared to the control and GGMOs. No significant changes between cadmium and GGMOs + cadmium variants in suberin lamellae development corresponded with any differences in cadmium uptake. GGMOs also stimulated Arabidopsis root growth under non-stress conditions. In this case, suberin lamellae were developed more distantly from the root apex in comparison with the control. Faster solute and water transport could explain the faster plant growth induced by GGMOs. Our results suggest that, in cadmium-stressed plants, GGMOs’ protective action is associated with the response at the metabolic level. © 2014 Elsevier GmbH. All rights reserved.
Introduction Heavy metal pollution is a serious and complex problem linking different environmental components. Soil and water contamination lead to plants growing in stressful conditions and accumulating toxic metals in their tissues (Clemens, 2006). This may cause human health problems (Järup and Åkesson, 2009), mostly as a consequence of the heavy metals entering the food chain. Among heavy metals, cadmium is the element of major concern, not only because of its widespread occurrence, but also because of its high toxicity and bioavailability for plants (Prokop et al., 2003). Considering the fact that phosphate fertilizers constitute a significant source of cadmium pollution (Chen et al., 2007), the level of cadmium is likely to be elevated in agricultural soils. Cadmium from soil is readily absorbed by roots, where it may cause growth inhibition and then be transported from roots to aerial plant parts (Clemens, 2006). Studies on cadmium uptake and distribution are essential to understanding the mechanisms involved in
∗ Corresponding author. Tel.: +421 2 59410265; fax: +421 2 59410222. E-mail addresses: [email protected], [email protected] (K. Kollárová). 0176-1617/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.12.012
cadmium tolerance, transportation and accumulation. When cadmium is absorbed by plants, it affects the water balance, photosynthetic apparatus and causes leaf chlorosis, oxidative stress, and the inhibition of stomata opening (Hasan et al., 2009). Root growth inhibition is one of the most visible and common symptoms of its phytotoxicity. This characteristic has been observed in almost all cadmium reports; however, more detailed studies in this field are rare. With regard to heavy metal toxicity, the development of approaches to clean up the soil, as well as strategies to decrease the accumulation and alleviate the damaging effects on plants, has become the object of interest (Sarwar et al., 2010). In addition to well-known, useful, but time-consuming methods such as breeding new and more resistant cultivars to be grown in contaminated soils, other approaches have also appeared. Preliminary studies are focused on the application of substances able to alleviate heavy metal stress. Among them, in the case of cadmium stress, are signaling molecules – salicylic acid (Belkadhi et al., 2008), polyamines (Hsu and Kao, 2007), plant nutrients (Sarwar et al., 2010), antioxidants – N-acetyl-l-cysteine (Deng et al., 2010), inorganic compounds – silicon (Vaculík et al., 2012), plant hormones (Munzuroglu and Zengin, 2006), and also biologically active oligosaccharides derived from bacteria (Ma et al., 2010).
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Biologically active oligosaccharides represent a group of regulatory molecules serving as signaling molecules in various processes of plant growth and development (Aldington et al., 1991; Ochoa-Villarreal et al., 2012) and playing a role in plantadaptive responses to abiotic and biotic stress (Van den Ende and Valluru, 2009; Zabotin et al., 2009). Our research is focused on a specific class of oligosaccharide-signaling molecules, galactoglucomannan oligosaccharides (GGMOs), derived from spruce galactoglucomannan (Karácsonyi et al., 1996; Capek et al., 2000). Galactoglucomannans are structural constituents of both the primary and secondary cell walls of higher plants (Kubaˇcková et al., 1992; Lundqvist et al., 2002). Galactoglucomannan polysaccharides may fulfill a role comparable to that of xyloglucans, impacting flexibility and forming growth-constraining networks with cellulose (Schröder et al., 2004). Galactoglucomannans have attracted a great deal of interest because of their possible applications as an oxygen barrier film in packaging materials, as a hydrogel in biomedical products and as an emulsion stabilizer in food and feed (Krawczyk and Jönsson, 2011). Zhao et al. (2013) found that a Populus endo-1,4--mannanase gene, PtrMAN6, which suppresses cell wall thickening during xylem differentiation, catalyzes the hydrolysis of mannan-type wall polysaccharides to produce GGMOs. GGMOs in turn serve as signaling molecules to regulate the transcriptional program of cell wall thickening. Exogenously added GGMOs regulate xylem cell differentiation ˇ (Benová-Kákoˇ sová et al., 2006; Richterová-Kuˇcerová et al., 2012; Kákoˇsová et al., 2013). GGMOs influence several other processes, such as elongation growth in various plants (Auxtová et al., 1995; Liˇsková et al., 1999; Kollárová et al., 2007, 2009; RichterováKuˇcerová et al., 2012), as well as cell division in Zinnia xylogenic ˇ sová et al., 2006), and regeneration of isoculture (Benová-Kákoˇ lated protoplasts (Kákoniová et al., 2010). Experimental evidence indicates that their action could be based on the interaction ˇ with auxin (Auxtová-Samajová et al., 1996). Exogenously applied GGMOs behave as auxin antagonists in both IBA (indole-3-butyric acid)-stimulated and -inhibited elongation growth (Kollárová et al., 2010). Their action is connected with changes in peroxidase activity (Kollárová et al., 2009, 2010). GGMOs are known as inhibitors of TNV virus infection (Slováková et al., 2000). They induce resistance manifested by the decrease of local lesions. Moreover, with the exception of plant growth and resistance, GGMOs are assumed to have a positive effect on human health because of their potential prebiotic activity (Willför et al., 2008; Polari et al., 2012). In this study, we hypothesized the potential of GGMOs to protect Arabidopsis plants against cadmium stress. The possible effects of GGMOs on root growth, root growth dynamics, and photosynthetic pigments content, as cadmium toxicity markers, were investigated. The second hypothesis was that GGMOs’ action in the presence of cadmium is connected with changes in cadmium accumulation, uptake or distribution in Arabidopsis plants.
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7/21 days. The length of the primary roots was determined after 7/21 days of cultivation. Preparation of galactoglucomannan oligosaccharides (GGMOs) GGMOs were obtained from spruce galactoglucomannan (composed of galactose, glucose and mannose in a 1:8:33 mol proportion) by partial acid hydrolysis, as described previously (Capek et al., 2000). The hydrolyzed mixture was separated on a column of Bio-Gel P2 into nine distinct fractions. Their degree of polymerization was identified by comparison with elution volumes of standard malto-oligosaccharides (Serva, Heidelberg, Germany). GGMOs (degree of polymerization 4–8) were combined and freeze-dried. Compositional analysis of the GGMOs revealed the presence of galactose (4.5%), glucose (21.1%), mannose (70.4%) and trace amounts of pentoses, xylose and arabinose. The oligosaccharides mixture was composed of tetramers (46%), pentamers (28%), hexamers (12%), heptamers (9%) and octamers (5%). Their number-average molecular mass (Mn ) was calculated to be 827. Primary root growth dynamics The primary root growth dynamics were measured by scoring the position of the root apex on the back of the Petri dish once per day (for the first 7 days). The root growth dynamics were determined according to Hlinková (1991). The growth index (RIj ) and growth dynamics (RI) were determined: RIj = (lj − l0 )/l0 , RIj = RIj − RIj−1 ; RIj – growth index over a certain time; j = 1, 2,. . .7 days; lj – length of the primary root on j-th day, l0 – length of the primary root at the beginning of the experiment. Determination of photosynthetic pigments Shoots of 21-day-old plants were collected (0.06 g of fresh weight per treatment) and photosynthetic pigments were extracted with 80% (v/v) acetone. Their concentrations were determined spectrophotometrically (Chl a at 663.2, Chl b at 646.8, and carotenoids at 470.0 nm), according to Lichtenthaler (1987). Chlorophyll and carotenoid absorption in the extract was measured using Libra S6 spectrophotometer (Biochrom Ltd, Cambridge). Inductively coupled plasma mass spectroscopy (ICP-MS) analysis
Methods
The 21-day-old plants were collected. Their roots and shoots were rinsed thoroughly with distilled water. The samples were oven-dried at 40 ◦ C for 7 days. Each sample was hydrolyzed in concentrated nitric acid in a PTFE pot of ZA-1 equipment. The samples were then boiled (160 ◦ C) for 5 h. Afterwards, refrigerated samples were supplemented with deionized water. The Cd content of each sample was determined using ICP-MS (Perkin Elmer Elan 6000), with 111 Cd and 114 Cd isotopes and Rh as inner standard.
Plant material and growth conditions
Cadmium staining
Surface decontaminated Arabidopsis thaliana (Ler) seeds were placed in Petri dishes on MS medium (Murashige and Skoog, 1962) supplemented with or without Cd(NO3 )2 (10−4 M), GGMOs (10−9 M), or GGMOs (10−10 M) + Cd(NO3 )2 (10−4 M). The most effective concentrations of GGMOs (chosen on the basis of previous experiments – data not shown) were used. Two days after stratification at 4 ◦ C in the dark, the dishes were transferred to a growth chamber at 24 ± 1 ◦ C, 60% relative humidity, under a 16 h photoperiod, at irradiance of 50–60 mol m−2 s−1 , in sterile conditions for
Cadmium staining in plant tissues (Cd and GGMOs + Cd treatment) was performed on days 1, 3, 5 and 7 of cultivation by histochemical techniques, according to Seregin and Ivanov (1997), with some modifications. In our work, whole plants were stained and vacuum infiltration of the dithizone solution (0.25 mg/ml) was used. To verify the accuracy of the method, control plants, as a negative control, were stained in every experiment. For positive control, 7-day-old plants of the cadmium hyperaccumulator Noccaea caerulescens (formerly Thlaspi), growing in the presence of
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30 25
control
GGMOs
Cd
GGMOs + Cd
Δ RI
20 15 10 5 0 1
2
3
4
5
6
7
Days
Fig. 2. Growth dynamics of Arabidopsis primary root elongation during 7 days of culture on media supplemented with GGMOs, Cd, or GGMOs + Cd. Control – MS medium without supplements.
Statistical analysis
Fig. 1. Arabidopsis primary root length after 7 days (A) and 21 days (B) of cultivation on media supplemented with GGMOs, Cd, or GGMOs + Cd. Control – MS medium without supplements. Different letters above bars mean significant differences between the treatments at 5% level according to LSD test.
The results of the growth parameters were analyzed using Statistica 9. The data were evaluated by analysis of variance (ANOVA) and comparisons between the mean values were made by at least significant difference (LSD) test at P < 0.05 and the standard error (SE) was calculated. In growth experiments, 50 plants per treatment, 100 plants per treatment in ICP-MS analysis, and 10–12 plants per treatment for microscopy, were analyzed. The data presented are from three independent experiments. Results and discussion
cadmium, were stained. All samples were observed using a Leica DMD 108 digital microscope. Determination of suberin lamellae formation Primary roots (after 21 days of cultivation) were covered with a thin layer of 4% agarose, embedded in agarose (6%), and then free-hand cross sections were prepared (Zelko et al., 2012). The sections were made in 1 mm intervals and stained with fluorol yellow 088 for the detection of suberin lamellae (Brundrett et al., 1991; Zelko et al., 2012). The sections were viewed and documented using an inverted microscope DMI3000 B Leica and digital camera DFC 295. Ultraviolet illumination was used for fluorescence microscopy (excitation filter TBP 400 + 495 + 570, beam splitter TFT 410 + 505 + 585, and emission filter TBP 460 + 530 + 610; the wavelengths are in nm). To overcome the problem of the different root lengths from each treatment, the distance of the suberin lamellae formation from the root apex was expressed as a percentage of the total root length (Vaculík et al., 2009; Redjala et al., 2011).
The root, as the first organ coming into contact with the cadmium, whose response is crucial to cope with Cd contamination, was the object of our study. Cadmium stress led to primary root growth inhibition (Fig. 1), as has long been known (Pál et al., 2006). Root elongation in Brassica as well as in Hordeum seedlings was inhibited by cadmium (Valentoviˇcová and Haluˇsková, 2010; Bauddh and Singh, 2011). The presence of GGMOs stimulated Arabidopsis root growth in non-stress conditions and diminished the negative effect of Cd on this process. Similarly, gibberellic acid and biologically active oligosaccharides derived from bacteria decreased the inhibiting effect of cadmium on barley root length (Munzuroglu and Zengin, 2006; Ma et al., 2010). For a more detailed view of the root growth rate, we calculated the root growth dynamics – the changes in root growth during the first 7 days of cultivation. Two peaks indicating accelerated growth were characteristic of the growth rate of control roots (Fig. 2); the first on the 4th and the second on the 6th day of cultivation. In the presence of GGMOs, the line was shifted to higher values and the first peak to the 3rd day of culture, indicating more
Fig. 3. Effects of Cd and GGMOs on chlorophyll a, chlorophyll >b, and carotenoids content (mg g−1 fresh weight) in Arabidopsis shoots after 21 days of culture on control medium, and on media supplemented with GGMOs, Cd, or GGMOs + Cd (A). Different letters above bars mean significant differences between the treatments at 5% level according to LSD test in certain photosynthetic pigments. Arabidopsis plants cultivated on media supplemented with GGMOs (C), Cd (D), or GGMOs + Cd (E). Control – MS medium without supplements (B). Scale bar is 2 mm.
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Table 1 Cadmium concentration in dry mass of root and shoot of Arabidopsis plants after 21 days of cultivation on medium with cadmium or GGMOs + cadmium. The same letters in rows mean non-significant differences between the treatments at 5% level according to LSD test. Cd concentration (mg kg−1 )
Cadmium
GGMOs+Cadmium
Root Shoot
1739 ± 92.1a 857 ± 35.5b
1696 ± 126.9a 937 ± 65.5b
accelerated growth compared to the control. The growth rate of primary roots in the presence of Cd was very slow and without distinct peaks. The growth dynamics of roots treated with GGMOs + Cd also showed two peaks, as in the case of GGMOs-treated roots, with the first acceleration on the 3rd day; however, the values were lower. The mitigation of cadmium-induced inhibition of root growth by GGMOs can be considered an important result, indicating the protective effect of these oligosaccharides. Cadmium induces complex changes in plants. In addition to reduction of tissue and organ growth, leaf chlorosis, and leaf and root necrosis represent well-known cadmium toxicity symptoms (Hernandez and Cooke, 1997). Because the photosynthetic pigment content is an important parameter that indicates Cd ´ ´ stress (Skórzynska-Polit and Baszynski, 1997; Gadapati and Macfie, 2006), we compared the chlorophyll and carotenoid content in the presence of Cd and GGMOs + Cd. In the Cd variant, a significant decrease was observed compared to control (Fig. 3). However, in the presence of GGMOs (GGMOs + Cd), the content of photosynthetic pigments increased significantly in comparison with Cd variant. A protective effect against chlorosis induced by cadmium has been reported for silicon (Vatehová et al., 2012), kinetin (Al-Hakimi, 2007), and salicylic acid (Krantev et al., 2008). Observing the Cd content can help clarify the protective effect of GGMOs. Basically, the protective substances can restrict the entry of pollutants or increase the tolerance to absorbed pollutants. In Cd and GGMOs + Cd-treated plants, there were no significant differences in cadmium concentrations in either roots or in shoots (Table 1). Similarly, IAA addition in heavy metal stress increased root growth in sunflower. However, root and shoot metal concentrations were not changed (Fässler et al., 2010). To understand the mechanisms involved in cadmium transport and storage, observation of cadmium distribution in a plant is essential. Studies on the distribution of metals within plant tissues are necessary in order to understand the physiology of metal hyperaccumulation and tolerance (Vogel-Mikuˇs et al., 2008). A study by Bezrukova et al. (2011) confirmed that the application of substances protecting plants against heavy metals can lead to changes in metal distribution. The chronological view of cadmium distribution in the tissues from the start of cultivation in this study is an original approach. In our study, cadmium distribution in tissues in dependency on the duration of Cd treatment and the presence of oligosaccharides was followed. Cadmium distribution in plant tissues was determined by the method of Seregin and Ivanov (1997), with some modifications for whole plants. To verify the accuracy of our modified method, the cadmium hyperaccumulator Noccaea caerulescens, growing in the presence of cadmium, was stained as a positive control. N. caerulescens was chosen on the basis of its relationship with Arabidopsis thaliana (Peer et al., 2003; Rigola et al., 2006). In N. caerulescens, the number of dithizone–cadmium complexes increased several times (Fig. 4A) in comparison with Arabidopsis (Fig. 4F), which was clearly demonstrated in rhizodermis and root hairs. Control plants, as a negative control, were stained in every experiment. We did not observe dithizone–cadmium complexes in control plants (Fig. 5A). The distribution of Cd in Arabidopsis tissues was dependent on the duration of the exposure (1, 3, 5, 7 days) to Cd
Fig. 4. Cadmium localization (orange or red complexes marked with arrows) in Noccaea caerulescens primary root (A) and in Arabidopsis primary root and hypocotyl after the first day of cadmium treatment (B, C), in Arabidopsis root hairs and hypocotyl after the third day of cadmium treatment (D, E), in Arabidopsis primary root and hypocotyl after the fifth day of cadmium treatment (F, G). Scale bar is 100 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
(Figs. 4B–G and 5B). After the first 24 h, cadmium was detected mostly in the root cap, in the apical region of the primary root (Fig. 4B) and also in the root-hypocotyl junction (Fig. 4C), but only in small amounts.
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Fig. 5. Cadmium localization (orange or red complexes marked with arrows) in Arabidopsis thaliana after the seventh day of cultivation. A – control variant (a – trichome, b – hypocotyl, c – primary root, d – root meristem), B – cadmium variant (a – trichome, b – hypocotyl xylem and xylem parenchyma cells, c – hypocotyl epidermis, d – primary and lateral root, e – root meristem), C – cadmium + GGMOs variant (a – trichome, b – hypocotyl epidermis, c – primary root, d – root meristem). Scale bar is 100 mm (A – a, c, d; B – a, d, e; C – a, b, c, d) and 50 mm (A – b; B – b, c). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
On day 3, Cd was detectable in the whole root, including root hairs (Fig. 4D) and xylem. Large dithizone–cadmium complexes were present in cells at the base of the hypocotyls (Fig. 4E). Moving further from the hypocotyl base, the complexes were smaller and situated close to the cell walls. Rarely were these complexes also found in xylem, xylem parenchyma cells or leaves, and then only in a few plants. After 5 days, the distribution in roots was similar; Cd absorbed by the root apex and root hairs was transported by the root (Fig. 4F – localization in xylem and xylem parenchymal cells) and hypocotyl xylem (Fig. 4G – localization in xylem and xylem parenchymal cells) to the petioles, and eventually to leaves. Cadmium was also present in lateral roots, especially at their base. On day 7, we identified cadmium in the primary root, mostly in xylem (Fig. 5Bd – localization in xylem and xylem parenchymal cells), at the base of lateral roots (Fig. 5Bd), and in the root meristem (Fig. 5Be). In Arabidopsis roots, Cd is localized mostly in vascular bundles (Isaure et al., 2006), but its presence has also been reported in all root tissues (Van Belleghem et al., 2007). In aboveground plant parts, there were many dithizone–cadmium complexes present in xylem (Fig. 5Bb – localization in xylem and xylem parenchymal cells) and in the center or close to the walls of epidermal (Fig. 5Bc) and primary cortical cells. In the leaves, cadmium was accumulated mostly in trichomes (Fig. 5Ba). It was also sporadically detected
between the guard cells in stomata. It can be speculated that the presence of dithizone–cadmium complexes in stomata is the consequence of the presence of cadmium in leaf apoplasm. However, further experimental evidence is required. Cadmium localization in Arabidopsis leaf trichomes has been reported by several authors (e.g. Ager et al., 2002; Isaure et al., 2006). The sequestration of Cd in these trichomes might be a way to protect the metabolically active cells from metal toxicity (Isaure et al., 2006). In the presence of GGMOs + Cd, there were not any significant changes in cadmium distribution in comparison with Cd-treated plants (Fig. 5B, C). On the other hand, the presence of WGA lectin changed cadmium localization. The decrease was observed in endodermis and inner tissues, which restricted the movement of Cd ions in the xylem vessels and reduced the level of their accumulation in the shoot (Bezrukova et al., 2011). To supplement our study with data reflecting cadmium uptake, the suberin lamellae development in primary root was ascertained. The suberin lamellae formation represents the second stage of endodermis development. Suberin deposition in the cell walls of the endodermis leads to a decrease in root radial conductivity (Hinsinger et al., 2009). In the context of solute transport regulation, suberin is the most important cell wall-impregnating substance (Soukup and Votrubová, 2002; Franke and Suberin, 2007). The response of plants to environmental conditions such as drought,
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Fig. 6. Scheme of suberin lamellae development in Arabidopsis primary root after 21 days of culture (A). Vertical axis represents the distance from the root apex expressed as percentage of total root length. The cross sections with suberin lamellae detection in primary root after 21 days of culture on control medium (B), on media supplemented with GGMOs (C), Cd (D), or GGMOs + Cd (E). Control – MS medium without supplements. The arrows indicate the layer in which suberin lamellae developed (C – without suberin lamellae). Scale bar is 25 m.
salt stress or oxygen deficiency is usually associated with suberin deposition in apoplasm. Observation of suberin lamellae formation in endodermis allows us to study Cd stress and GGMOs action from the viewpoint of Cd uptake. In Cd-treated plants, the formation of suberin lamellae (Fig. 6A and D) was shifted closer to the root apex in comparison with the control (34% in Cd, 62% in the control). Several authors have confirmed that accelerated formation of suberin lamellae is associated with reduced cadmium uptake (Redjala et al., 2011; Vatehová et al., 2012). The suberin lamellae in GGMOs + Cd-treated plants (Fig. 6A and E) were developed in 43% of primary root length. However, the statistical difference between Cd and GGMOs + Cd values was not significant. This finding is in concordance with the fact that we did not observe changes in cadmium accumulation between these variants. In plants growing in the presence of GGMOs, the suberin lamellae developed more distantly from the root apex (62% in the control, 90% in GGMOs treatment) (Fig. 6A–C). The relative delay of suberin lamellae formation in GGMOs treatment can be attributed to the root growth-stimulating effect of GGMOs. Based on the fact that suberized walls in the endodermis are the major barriers to radial ion and water movements in plant roots (Reinhardt and Rost, 1995), the delay in suberin lamellae formation in GGMOs-treated plants could contribute to faster transport of water and solutes, facilitating faster growth. GGMOs proved to be effective against cadmium stress in Arabidopsis plants. They diminished chosen cadmium toxicity symptoms. Because GGMOs did not influence cadmium concentration, distribution in plant tissues or uptake, it can be assumed that the protective role of these oligosaccharides is based on an increased tolerance to absorbed cadmium at a metabolic level. Acknowledgements This work was supported by grants from the Slovak Grant Agency for Science (VEGA no. 2/0046/10 and the COST Action FA0905.
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Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Physiology
Galactoglucomannan oligosaccharides alleviate cadmium stress in Arabidopsis Danica Kuˇcerová a,b , Karin Kollárová a,∗ , Ivan Zelko a , Zuzana Vatehová a , Desana Liˇsková a a b
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovakia Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, 845 23 Bratislava, Slovakia
a r t i c l e
i n f o
Article history: Received 8 July 2013 Received in revised form 26 November 2013 Accepted 10 December 2013 Available online 13 March 2014 Keywords: Arabidopsis thaliana Cadmium distribution in tissues Galactoglucomannan oligosaccharides Suberin lamellae development
s u m m a r y Our study focused on the mediatory role of galactoglucomannan oligosaccharides (GGMOs) in plant protection against cadmium stress, examined mainly on the primary root growth of Arabidopsis thaliana. The application of GGMOs diminished the negative effect of cadmium on root length, root growth dynamics and also on photosynthetic pigment content. We tested the hypothesis that the effect of GGMOs is associated with decreased cadmium accumulation or its modified distribution. Cadmium distribution was observed chronologically from the first day of plant culture and depended on the duration of cadmium treatment. First, cadmium was stored in the root and hypocotyl and later transported by xylem to the leaves and stored there in trichomes. The protective effect of GGMOs was not based on modified cadmium distribution or its decreased accumulation. In cadmium and GGMOs + cadmiumtreated plants, the formation of suberin lamellae was shifted closer to the root apex compared to the control and GGMOs. No significant changes between cadmium and GGMOs + cadmium variants in suberin lamellae development corresponded with any differences in cadmium uptake. GGMOs also stimulated Arabidopsis root growth under non-stress conditions. In this case, suberin lamellae were developed more distantly from the root apex in comparison with the control. Faster solute and water transport could explain the faster plant growth induced by GGMOs. Our results suggest that, in cadmium-stressed plants, GGMOs’ protective action is associated with the response at the metabolic level. © 2014 Elsevier GmbH. All rights reserved.
Introduction Heavy metal pollution is a serious and complex problem linking different environmental components. Soil and water contamination lead to plants growing in stressful conditions and accumulating toxic metals in their tissues (Clemens, 2006). This may cause human health problems (Järup and Åkesson, 2009), mostly as a consequence of the heavy metals entering the food chain. Among heavy metals, cadmium is the element of major concern, not only because of its widespread occurrence, but also because of its high toxicity and bioavailability for plants (Prokop et al., 2003). Considering the fact that phosphate fertilizers constitute a significant source of cadmium pollution (Chen et al., 2007), the level of cadmium is likely to be elevated in agricultural soils. Cadmium from soil is readily absorbed by roots, where it may cause growth inhibition and then be transported from roots to aerial plant parts (Clemens, 2006). Studies on cadmium uptake and distribution are essential to understanding the mechanisms involved in
∗ Corresponding author. Tel.: +421 2 59410265; fax: +421 2 59410222. E-mail addresses: [email protected], [email protected] (K. Kollárová). 0176-1617/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.12.012
cadmium tolerance, transportation and accumulation. When cadmium is absorbed by plants, it affects the water balance, photosynthetic apparatus and causes leaf chlorosis, oxidative stress, and the inhibition of stomata opening (Hasan et al., 2009). Root growth inhibition is one of the most visible and common symptoms of its phytotoxicity. This characteristic has been observed in almost all cadmium reports; however, more detailed studies in this field are rare. With regard to heavy metal toxicity, the development of approaches to clean up the soil, as well as strategies to decrease the accumulation and alleviate the damaging effects on plants, has become the object of interest (Sarwar et al., 2010). In addition to well-known, useful, but time-consuming methods such as breeding new and more resistant cultivars to be grown in contaminated soils, other approaches have also appeared. Preliminary studies are focused on the application of substances able to alleviate heavy metal stress. Among them, in the case of cadmium stress, are signaling molecules – salicylic acid (Belkadhi et al., 2008), polyamines (Hsu and Kao, 2007), plant nutrients (Sarwar et al., 2010), antioxidants – N-acetyl-l-cysteine (Deng et al., 2010), inorganic compounds – silicon (Vaculík et al., 2012), plant hormones (Munzuroglu and Zengin, 2006), and also biologically active oligosaccharides derived from bacteria (Ma et al., 2010).
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Biologically active oligosaccharides represent a group of regulatory molecules serving as signaling molecules in various processes of plant growth and development (Aldington et al., 1991; Ochoa-Villarreal et al., 2012) and playing a role in plantadaptive responses to abiotic and biotic stress (Van den Ende and Valluru, 2009; Zabotin et al., 2009). Our research is focused on a specific class of oligosaccharide-signaling molecules, galactoglucomannan oligosaccharides (GGMOs), derived from spruce galactoglucomannan (Karácsonyi et al., 1996; Capek et al., 2000). Galactoglucomannans are structural constituents of both the primary and secondary cell walls of higher plants (Kubaˇcková et al., 1992; Lundqvist et al., 2002). Galactoglucomannan polysaccharides may fulfill a role comparable to that of xyloglucans, impacting flexibility and forming growth-constraining networks with cellulose (Schröder et al., 2004). Galactoglucomannans have attracted a great deal of interest because of their possible applications as an oxygen barrier film in packaging materials, as a hydrogel in biomedical products and as an emulsion stabilizer in food and feed (Krawczyk and Jönsson, 2011). Zhao et al. (2013) found that a Populus endo-1,4--mannanase gene, PtrMAN6, which suppresses cell wall thickening during xylem differentiation, catalyzes the hydrolysis of mannan-type wall polysaccharides to produce GGMOs. GGMOs in turn serve as signaling molecules to regulate the transcriptional program of cell wall thickening. Exogenously added GGMOs regulate xylem cell differentiation ˇ (Benová-Kákoˇ sová et al., 2006; Richterová-Kuˇcerová et al., 2012; Kákoˇsová et al., 2013). GGMOs influence several other processes, such as elongation growth in various plants (Auxtová et al., 1995; Liˇsková et al., 1999; Kollárová et al., 2007, 2009; RichterováKuˇcerová et al., 2012), as well as cell division in Zinnia xylogenic ˇ sová et al., 2006), and regeneration of isoculture (Benová-Kákoˇ lated protoplasts (Kákoniová et al., 2010). Experimental evidence indicates that their action could be based on the interaction ˇ with auxin (Auxtová-Samajová et al., 1996). Exogenously applied GGMOs behave as auxin antagonists in both IBA (indole-3-butyric acid)-stimulated and -inhibited elongation growth (Kollárová et al., 2010). Their action is connected with changes in peroxidase activity (Kollárová et al., 2009, 2010). GGMOs are known as inhibitors of TNV virus infection (Slováková et al., 2000). They induce resistance manifested by the decrease of local lesions. Moreover, with the exception of plant growth and resistance, GGMOs are assumed to have a positive effect on human health because of their potential prebiotic activity (Willför et al., 2008; Polari et al., 2012). In this study, we hypothesized the potential of GGMOs to protect Arabidopsis plants against cadmium stress. The possible effects of GGMOs on root growth, root growth dynamics, and photosynthetic pigments content, as cadmium toxicity markers, were investigated. The second hypothesis was that GGMOs’ action in the presence of cadmium is connected with changes in cadmium accumulation, uptake or distribution in Arabidopsis plants.
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7/21 days. The length of the primary roots was determined after 7/21 days of cultivation. Preparation of galactoglucomannan oligosaccharides (GGMOs) GGMOs were obtained from spruce galactoglucomannan (composed of galactose, glucose and mannose in a 1:8:33 mol proportion) by partial acid hydrolysis, as described previously (Capek et al., 2000). The hydrolyzed mixture was separated on a column of Bio-Gel P2 into nine distinct fractions. Their degree of polymerization was identified by comparison with elution volumes of standard malto-oligosaccharides (Serva, Heidelberg, Germany). GGMOs (degree of polymerization 4–8) were combined and freeze-dried. Compositional analysis of the GGMOs revealed the presence of galactose (4.5%), glucose (21.1%), mannose (70.4%) and trace amounts of pentoses, xylose and arabinose. The oligosaccharides mixture was composed of tetramers (46%), pentamers (28%), hexamers (12%), heptamers (9%) and octamers (5%). Their number-average molecular mass (Mn ) was calculated to be 827. Primary root growth dynamics The primary root growth dynamics were measured by scoring the position of the root apex on the back of the Petri dish once per day (for the first 7 days). The root growth dynamics were determined according to Hlinková (1991). The growth index (RIj ) and growth dynamics (RI) were determined: RIj = (lj − l0 )/l0 , RIj = RIj − RIj−1 ; RIj – growth index over a certain time; j = 1, 2,. . .7 days; lj – length of the primary root on j-th day, l0 – length of the primary root at the beginning of the experiment. Determination of photosynthetic pigments Shoots of 21-day-old plants were collected (0.06 g of fresh weight per treatment) and photosynthetic pigments were extracted with 80% (v/v) acetone. Their concentrations were determined spectrophotometrically (Chl a at 663.2, Chl b at 646.8, and carotenoids at 470.0 nm), according to Lichtenthaler (1987). Chlorophyll and carotenoid absorption in the extract was measured using Libra S6 spectrophotometer (Biochrom Ltd, Cambridge). Inductively coupled plasma mass spectroscopy (ICP-MS) analysis
Methods
The 21-day-old plants were collected. Their roots and shoots were rinsed thoroughly with distilled water. The samples were oven-dried at 40 ◦ C for 7 days. Each sample was hydrolyzed in concentrated nitric acid in a PTFE pot of ZA-1 equipment. The samples were then boiled (160 ◦ C) for 5 h. Afterwards, refrigerated samples were supplemented with deionized water. The Cd content of each sample was determined using ICP-MS (Perkin Elmer Elan 6000), with 111 Cd and 114 Cd isotopes and Rh as inner standard.
Plant material and growth conditions
Cadmium staining
Surface decontaminated Arabidopsis thaliana (Ler) seeds were placed in Petri dishes on MS medium (Murashige and Skoog, 1962) supplemented with or without Cd(NO3 )2 (10−4 M), GGMOs (10−9 M), or GGMOs (10−10 M) + Cd(NO3 )2 (10−4 M). The most effective concentrations of GGMOs (chosen on the basis of previous experiments – data not shown) were used. Two days after stratification at 4 ◦ C in the dark, the dishes were transferred to a growth chamber at 24 ± 1 ◦ C, 60% relative humidity, under a 16 h photoperiod, at irradiance of 50–60 mol m−2 s−1 , in sterile conditions for
Cadmium staining in plant tissues (Cd and GGMOs + Cd treatment) was performed on days 1, 3, 5 and 7 of cultivation by histochemical techniques, according to Seregin and Ivanov (1997), with some modifications. In our work, whole plants were stained and vacuum infiltration of the dithizone solution (0.25 mg/ml) was used. To verify the accuracy of the method, control plants, as a negative control, were stained in every experiment. For positive control, 7-day-old plants of the cadmium hyperaccumulator Noccaea caerulescens (formerly Thlaspi), growing in the presence of
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30 25
control
GGMOs
Cd
GGMOs + Cd
Δ RI
20 15 10 5 0 1
2
3
4
5
6
7
Days
Fig. 2. Growth dynamics of Arabidopsis primary root elongation during 7 days of culture on media supplemented with GGMOs, Cd, or GGMOs + Cd. Control – MS medium without supplements.
Statistical analysis
Fig. 1. Arabidopsis primary root length after 7 days (A) and 21 days (B) of cultivation on media supplemented with GGMOs, Cd, or GGMOs + Cd. Control – MS medium without supplements. Different letters above bars mean significant differences between the treatments at 5% level according to LSD test.
The results of the growth parameters were analyzed using Statistica 9. The data were evaluated by analysis of variance (ANOVA) and comparisons between the mean values were made by at least significant difference (LSD) test at P < 0.05 and the standard error (SE) was calculated. In growth experiments, 50 plants per treatment, 100 plants per treatment in ICP-MS analysis, and 10–12 plants per treatment for microscopy, were analyzed. The data presented are from three independent experiments. Results and discussion
cadmium, were stained. All samples were observed using a Leica DMD 108 digital microscope. Determination of suberin lamellae formation Primary roots (after 21 days of cultivation) were covered with a thin layer of 4% agarose, embedded in agarose (6%), and then free-hand cross sections were prepared (Zelko et al., 2012). The sections were made in 1 mm intervals and stained with fluorol yellow 088 for the detection of suberin lamellae (Brundrett et al., 1991; Zelko et al., 2012). The sections were viewed and documented using an inverted microscope DMI3000 B Leica and digital camera DFC 295. Ultraviolet illumination was used for fluorescence microscopy (excitation filter TBP 400 + 495 + 570, beam splitter TFT 410 + 505 + 585, and emission filter TBP 460 + 530 + 610; the wavelengths are in nm). To overcome the problem of the different root lengths from each treatment, the distance of the suberin lamellae formation from the root apex was expressed as a percentage of the total root length (Vaculík et al., 2009; Redjala et al., 2011).
The root, as the first organ coming into contact with the cadmium, whose response is crucial to cope with Cd contamination, was the object of our study. Cadmium stress led to primary root growth inhibition (Fig. 1), as has long been known (Pál et al., 2006). Root elongation in Brassica as well as in Hordeum seedlings was inhibited by cadmium (Valentoviˇcová and Haluˇsková, 2010; Bauddh and Singh, 2011). The presence of GGMOs stimulated Arabidopsis root growth in non-stress conditions and diminished the negative effect of Cd on this process. Similarly, gibberellic acid and biologically active oligosaccharides derived from bacteria decreased the inhibiting effect of cadmium on barley root length (Munzuroglu and Zengin, 2006; Ma et al., 2010). For a more detailed view of the root growth rate, we calculated the root growth dynamics – the changes in root growth during the first 7 days of cultivation. Two peaks indicating accelerated growth were characteristic of the growth rate of control roots (Fig. 2); the first on the 4th and the second on the 6th day of cultivation. In the presence of GGMOs, the line was shifted to higher values and the first peak to the 3rd day of culture, indicating more
Fig. 3. Effects of Cd and GGMOs on chlorophyll a, chlorophyll >b, and carotenoids content (mg g−1 fresh weight) in Arabidopsis shoots after 21 days of culture on control medium, and on media supplemented with GGMOs, Cd, or GGMOs + Cd (A). Different letters above bars mean significant differences between the treatments at 5% level according to LSD test in certain photosynthetic pigments. Arabidopsis plants cultivated on media supplemented with GGMOs (C), Cd (D), or GGMOs + Cd (E). Control – MS medium without supplements (B). Scale bar is 2 mm.
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Table 1 Cadmium concentration in dry mass of root and shoot of Arabidopsis plants after 21 days of cultivation on medium with cadmium or GGMOs + cadmium. The same letters in rows mean non-significant differences between the treatments at 5% level according to LSD test. Cd concentration (mg kg−1 )
Cadmium
GGMOs+Cadmium
Root Shoot
1739 ± 92.1a 857 ± 35.5b
1696 ± 126.9a 937 ± 65.5b
accelerated growth compared to the control. The growth rate of primary roots in the presence of Cd was very slow and without distinct peaks. The growth dynamics of roots treated with GGMOs + Cd also showed two peaks, as in the case of GGMOs-treated roots, with the first acceleration on the 3rd day; however, the values were lower. The mitigation of cadmium-induced inhibition of root growth by GGMOs can be considered an important result, indicating the protective effect of these oligosaccharides. Cadmium induces complex changes in plants. In addition to reduction of tissue and organ growth, leaf chlorosis, and leaf and root necrosis represent well-known cadmium toxicity symptoms (Hernandez and Cooke, 1997). Because the photosynthetic pigment content is an important parameter that indicates Cd ´ ´ stress (Skórzynska-Polit and Baszynski, 1997; Gadapati and Macfie, 2006), we compared the chlorophyll and carotenoid content in the presence of Cd and GGMOs + Cd. In the Cd variant, a significant decrease was observed compared to control (Fig. 3). However, in the presence of GGMOs (GGMOs + Cd), the content of photosynthetic pigments increased significantly in comparison with Cd variant. A protective effect against chlorosis induced by cadmium has been reported for silicon (Vatehová et al., 2012), kinetin (Al-Hakimi, 2007), and salicylic acid (Krantev et al., 2008). Observing the Cd content can help clarify the protective effect of GGMOs. Basically, the protective substances can restrict the entry of pollutants or increase the tolerance to absorbed pollutants. In Cd and GGMOs + Cd-treated plants, there were no significant differences in cadmium concentrations in either roots or in shoots (Table 1). Similarly, IAA addition in heavy metal stress increased root growth in sunflower. However, root and shoot metal concentrations were not changed (Fässler et al., 2010). To understand the mechanisms involved in cadmium transport and storage, observation of cadmium distribution in a plant is essential. Studies on the distribution of metals within plant tissues are necessary in order to understand the physiology of metal hyperaccumulation and tolerance (Vogel-Mikuˇs et al., 2008). A study by Bezrukova et al. (2011) confirmed that the application of substances protecting plants against heavy metals can lead to changes in metal distribution. The chronological view of cadmium distribution in the tissues from the start of cultivation in this study is an original approach. In our study, cadmium distribution in tissues in dependency on the duration of Cd treatment and the presence of oligosaccharides was followed. Cadmium distribution in plant tissues was determined by the method of Seregin and Ivanov (1997), with some modifications for whole plants. To verify the accuracy of our modified method, the cadmium hyperaccumulator Noccaea caerulescens, growing in the presence of cadmium, was stained as a positive control. N. caerulescens was chosen on the basis of its relationship with Arabidopsis thaliana (Peer et al., 2003; Rigola et al., 2006). In N. caerulescens, the number of dithizone–cadmium complexes increased several times (Fig. 4A) in comparison with Arabidopsis (Fig. 4F), which was clearly demonstrated in rhizodermis and root hairs. Control plants, as a negative control, were stained in every experiment. We did not observe dithizone–cadmium complexes in control plants (Fig. 5A). The distribution of Cd in Arabidopsis tissues was dependent on the duration of the exposure (1, 3, 5, 7 days) to Cd
Fig. 4. Cadmium localization (orange or red complexes marked with arrows) in Noccaea caerulescens primary root (A) and in Arabidopsis primary root and hypocotyl after the first day of cadmium treatment (B, C), in Arabidopsis root hairs and hypocotyl after the third day of cadmium treatment (D, E), in Arabidopsis primary root and hypocotyl after the fifth day of cadmium treatment (F, G). Scale bar is 100 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
(Figs. 4B–G and 5B). After the first 24 h, cadmium was detected mostly in the root cap, in the apical region of the primary root (Fig. 4B) and also in the root-hypocotyl junction (Fig. 4C), but only in small amounts.
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Fig. 5. Cadmium localization (orange or red complexes marked with arrows) in Arabidopsis thaliana after the seventh day of cultivation. A – control variant (a – trichome, b – hypocotyl, c – primary root, d – root meristem), B – cadmium variant (a – trichome, b – hypocotyl xylem and xylem parenchyma cells, c – hypocotyl epidermis, d – primary and lateral root, e – root meristem), C – cadmium + GGMOs variant (a – trichome, b – hypocotyl epidermis, c – primary root, d – root meristem). Scale bar is 100 mm (A – a, c, d; B – a, d, e; C – a, b, c, d) and 50 mm (A – b; B – b, c). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
On day 3, Cd was detectable in the whole root, including root hairs (Fig. 4D) and xylem. Large dithizone–cadmium complexes were present in cells at the base of the hypocotyls (Fig. 4E). Moving further from the hypocotyl base, the complexes were smaller and situated close to the cell walls. Rarely were these complexes also found in xylem, xylem parenchyma cells or leaves, and then only in a few plants. After 5 days, the distribution in roots was similar; Cd absorbed by the root apex and root hairs was transported by the root (Fig. 4F – localization in xylem and xylem parenchymal cells) and hypocotyl xylem (Fig. 4G – localization in xylem and xylem parenchymal cells) to the petioles, and eventually to leaves. Cadmium was also present in lateral roots, especially at their base. On day 7, we identified cadmium in the primary root, mostly in xylem (Fig. 5Bd – localization in xylem and xylem parenchymal cells), at the base of lateral roots (Fig. 5Bd), and in the root meristem (Fig. 5Be). In Arabidopsis roots, Cd is localized mostly in vascular bundles (Isaure et al., 2006), but its presence has also been reported in all root tissues (Van Belleghem et al., 2007). In aboveground plant parts, there were many dithizone–cadmium complexes present in xylem (Fig. 5Bb – localization in xylem and xylem parenchymal cells) and in the center or close to the walls of epidermal (Fig. 5Bc) and primary cortical cells. In the leaves, cadmium was accumulated mostly in trichomes (Fig. 5Ba). It was also sporadically detected
between the guard cells in stomata. It can be speculated that the presence of dithizone–cadmium complexes in stomata is the consequence of the presence of cadmium in leaf apoplasm. However, further experimental evidence is required. Cadmium localization in Arabidopsis leaf trichomes has been reported by several authors (e.g. Ager et al., 2002; Isaure et al., 2006). The sequestration of Cd in these trichomes might be a way to protect the metabolically active cells from metal toxicity (Isaure et al., 2006). In the presence of GGMOs + Cd, there were not any significant changes in cadmium distribution in comparison with Cd-treated plants (Fig. 5B, C). On the other hand, the presence of WGA lectin changed cadmium localization. The decrease was observed in endodermis and inner tissues, which restricted the movement of Cd ions in the xylem vessels and reduced the level of their accumulation in the shoot (Bezrukova et al., 2011). To supplement our study with data reflecting cadmium uptake, the suberin lamellae development in primary root was ascertained. The suberin lamellae formation represents the second stage of endodermis development. Suberin deposition in the cell walls of the endodermis leads to a decrease in root radial conductivity (Hinsinger et al., 2009). In the context of solute transport regulation, suberin is the most important cell wall-impregnating substance (Soukup and Votrubová, 2002; Franke and Suberin, 2007). The response of plants to environmental conditions such as drought,
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Fig. 6. Scheme of suberin lamellae development in Arabidopsis primary root after 21 days of culture (A). Vertical axis represents the distance from the root apex expressed as percentage of total root length. The cross sections with suberin lamellae detection in primary root after 21 days of culture on control medium (B), on media supplemented with GGMOs (C), Cd (D), or GGMOs + Cd (E). Control – MS medium without supplements. The arrows indicate the layer in which suberin lamellae developed (C – without suberin lamellae). Scale bar is 25 m.
salt stress or oxygen deficiency is usually associated with suberin deposition in apoplasm. Observation of suberin lamellae formation in endodermis allows us to study Cd stress and GGMOs action from the viewpoint of Cd uptake. In Cd-treated plants, the formation of suberin lamellae (Fig. 6A and D) was shifted closer to the root apex in comparison with the control (34% in Cd, 62% in the control). Several authors have confirmed that accelerated formation of suberin lamellae is associated with reduced cadmium uptake (Redjala et al., 2011; Vatehová et al., 2012). The suberin lamellae in GGMOs + Cd-treated plants (Fig. 6A and E) were developed in 43% of primary root length. However, the statistical difference between Cd and GGMOs + Cd values was not significant. This finding is in concordance with the fact that we did not observe changes in cadmium accumulation between these variants. In plants growing in the presence of GGMOs, the suberin lamellae developed more distantly from the root apex (62% in the control, 90% in GGMOs treatment) (Fig. 6A–C). The relative delay of suberin lamellae formation in GGMOs treatment can be attributed to the root growth-stimulating effect of GGMOs. Based on the fact that suberized walls in the endodermis are the major barriers to radial ion and water movements in plant roots (Reinhardt and Rost, 1995), the delay in suberin lamellae formation in GGMOs-treated plants could contribute to faster transport of water and solutes, facilitating faster growth. GGMOs proved to be effective against cadmium stress in Arabidopsis plants. They diminished chosen cadmium toxicity symptoms. Because GGMOs did not influence cadmium concentration, distribution in plant tissues or uptake, it can be assumed that the protective role of these oligosaccharides is based on an increased tolerance to absorbed cadmium at a metabolic level. Acknowledgements This work was supported by grants from the Slovak Grant Agency for Science (VEGA no. 2/0046/10 and the COST Action FA0905.
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