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Phosphorus deficiency-induced reduction in root hydraulic conductivity in Medicago falcata is associated with ethylene production
Environmental and Experimental Botany 67 (2009) 172–177
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Phosphorus deficiency-induced reduction in root hydraulic conductivity in Medicago falcata is associated with ethylene production Yan-Su Li, Xiao-Tao Mao, Qiu-Ying Tian, Ling-Hao Li, Wen-Hao Zhang ∗ State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China
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Article history: Received 13 January 2009 Received in revised form 26 May 2009 Accepted 28 May 2009 Keywords: Phosphorus deficiency Root hydraulic conductivity Ethylene production Aquaporins Medicago falcata L
a b s t r a c t Plants grown in phosphorus-deficient solutions often exhibit disruption of water transport due to reduction in root hydraulic conductivity (Lpr) and enhanced ethylene production. To uncover the relationship between the reduction in Lpr and increase in ethylene production, we investigated effect of phosphorus (P) deficiency on ethylene production and Lpr in legume plants of Medicago falcata L. There was an increase in ethylene production and a reduction of Lpr of M. falcata roots when M. falcata seedlings grown in P sufficient solutions (0.5 mM H2 PO4 2− ) were transferred to P-deficient solutions (5 M H2 PO4 2− ). Antagonists of ethylene biosynthesis, CoCl2 and aminoethoxyvinylglycine (AVG), abolished the P deficiency-induced ethylene production. Root hydraulic conductivity of M. falcata seedlings grown in P-sufficient solutions was insensitive to CoCl2 and AVG, while the two chemicals enhanced Lpr for those grown in P-deficient solutions, suggesting that P deficiency-induced decrease in Lpr can be reversed by inhibiting ethylene production. Ethylene precursor 1-amino cyclopropane-1-carboxylic acid (ACC) and ethylene donor ethephon had greater inhibitory effect on Lpr of P-sufficient seedlings than that of P-deficient seedlings. Root hydraulic conductivity of P-sufficient seedlings was more sensitive to HgCl2 than that of P-deficient seedlings. Taken together, these findings suggest that ethylene induced by P deficiency may play an important role in modulation of root hydraulic conductivity by affecting aquaporins in plants. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Root hydraulic conductivity (Lpr), which mainly reflects the hydraulic resistance for radial water flow from rhizosphere to the root xylem, is a key parameter in regulation of plant water status by modulating water transport in roots (Steudle and Peterson, 1998). Root hydraulic conductivity has been shown to be sensitive to a number of environmental factors. These include hypoxia (Zhang and Tyerman, 1991; Birner and Steudle, 1993; Zhang and Tyerman, 1999), salinity (Carvajal et al., 1999; Lopez-Berenguer et al., 2006) and low temperature (Wan et al., 1999; Lee et al., 2005; Murai-Hatano et al., 2008). In plants, water flow across roots can occur along a combination of apoplastic, symplastic, and transcellular pathways (Steudle and Peterson, 1998). The dominant water transport pathways in roots are dependent on factors such as plant species, growth conditions, developmental stages and driving force for radial water flow across roots (Steudle and Peterson, 1998). The
Abbreviations: ACS, 1-aminocyclopropane-1-carboxylic acid synthease; ACO, 1aminocyclopropane-1-carboxylic acid oxidase; AVG, aminoethoxyvinylglycine; DTT, dithiothreitol; Eth, ethephone; Jv, steady-state flow rate; Lpr, root hydraulic conductivity. ∗ Corresponding author. Tel.: +86 10 6283 6697; fax: +86 10 6259 2430. E-mail address: [email protected] (W.-H. Zhang). 0098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2009.05.013
discovery of aquaporins as major proteins to facilitate water transport across membranes greatly advances our knowledge on plant water transport at both cellular and whole plant levels (Javot and Maurel, 2002; Tyerman et al., 2002). In this context, the changes in Lpr in response to abiotic stresses have frequently been ascribed to reductions in activities of aquaporins as demonstrated by antagonist of aquaporins and expression of genes encoding aqauporins (Martinez-Ballesta et al., 2003; Murai-Hatano et al., 2008). Like ion channels, recent studies have revealed that aquaporins can also be gated (opening and closing) by factors such as cytoplasmic pH and free Ca2+ activity (Tournaire-Roux et al., 2003; Alleva et al., 2006), mechanical stimuli (Wan et al., 2004) and phytohormones (Hose et al., 2000; Wan et al., 2004). Therefore, plants can respond to abiotic stress by regulating their Lpr via changes in aquaporin activity. There have been a number of reports demonstrating that Lpr is reduced in response to deficiency in anion nutrients such as phosphate (Radin and Eidenbock, 1984; Radin and Matthews, 1989; Carvajal et al., 1996; Clarkson et al., 2000; Fan et al., 2007) nitrate (Radin and Matthews, 1989; Carvajal et al., 1996; Clarkson et al., 2000) and sulfate (Karmoker et al., 1991; Clarkson et al., 2000) in different plant species. Root hydraulic conductivity is also sensitive to plant hormones. For instance, there are reports that Lpr is modulated by abscisic acid (ABA) (Hose et al., 2000; Wan et al., 2004) and ethylene (Kamaluddin and Zwiazek, 2002). In addition to changes in Lpr, P deficiency also induces ethylene production
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(Lynch and Brown, 1997; Borch et al., 1999; Schmidt, 2001). The P deficiency-induced ethylene production may be involved in modulation of root growth and development (Borch et al., 1999; Ma et al., 2003; Zhang et al., 2003). However, there has been no study to examine the relationship between P deficiency-induced ethylene production and reduction in Lpr under conditions of P deficiency. In the present study, we investigated effect of P deficiency on Lpr of legume plants of Medicago falcata, an important native legume plant widely distributed in Inner Mongolia grassland in northern China, and evaluated the role of ethylene in the changes in Lpr induced by P deficiency. 2. Materials and methods 2.1. Plant material Seeds of M. falcata L., which were obtained from the Institute of Grassland Research, the Chinese Academy of Agricultural Sciences, were dipped in sulfuric acid for approx. 5 min to destroy the seed capsule, and then rinsed thoroughly with sterilized water. Thereafter the seeds were spread on 0.75% agar to germinate at 25 ◦ C till the radicals being about 2 cm. The seedlings were transferred to plastic buckets filled with 5 L fully aerated nutrient solution (Barker et al., 2006). Each bucket contained 15 plants. Plants were grown in a growth chamber with 25 ◦ C (day)/20 ◦ C (night), 80% relative air humidity, 200–230 mol m−2 s−2 photosynthetic photon flux density and 16/8 h day–night period. After grown in the culture solution for 3 weeks, half of the plants were transferred to P-deficient (−P) solutions. Potassium phosphate was added to give a final concentration of 500 M of H2 PO4 − for P-sufficient (+P) and 5 M for P-deficient (−P) media, respectively. To maintain the identical concentrations of K between P-sufficient and P-deficient solutions, 455 M KCl was added in the P-deficient solutions. Other components of the hydroponic nutrient solution were: 1 mM MgSO4 , 0.25 mM K2 SO4 , 0.25 mM CaCl2 , 1 mM NH4 NO3 , 2.5 mM KNO3 , 100 M Fe–Na–EDTA, 30 M H3 BO3 , 5 M MnSO4 , 1 M ZnSO4 , 1 M CuSO4 , 0.7 M Na2 MoO4 and 50 M KCl. pH of the hydroponic solutions was adjusted to 6.0.
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Instruments, Corvallis, OR) and filled with the solution used for culturing plants. The de-topped root system of M. falcata seedlings grown in P-sufficient and P-deficient solutions in the absence or presence of various chemicals (AVG, CoCl2 , ACC and ethephone) was immediately sealed in the pressure chamber. An appropriate pressure was gradually applied with compressed air and maintained during the measurements. The exuded xylem sap from the detoped root system was continuously collected and weighed. Root flow rates of whole root systems were monitored for linearity for at least 30 min. The flow rate was plotted against the applied pressure between 0.2 and 0.8 MPa, and the resultant slope (K) of the curve was then used to calculate hydraulic conductivity after determining surface area (A) of roots by scanning the root system with a scanner and analyzing the root images by Image J software (Kimura and Yamasaki, 2003). Hydraulic conductivity of the whole root system (Lpr) was calculated by: Lpr = K/A. 3. Results 3.1. Exposure of M. falcata seedlings to P-deficient solution led to evolution of ethylene To test whether P deficiency affects ethylene production from roots of M. falcata, ethylene production in response to P deficiency was studied. As shown in Fig. 1A, a marked increase in ethylene evolution from M. falcata roots was observed when M. falcata seedlings were transferred from P sufficient (500 M) to P deficient (5 M) solutions for 2d. For instance, ethylene evolution from P sufficient roots was 0.20 ± 0.09 ppm g−1 FW h−1 , while this evolution was
2.2. Determination of ethylene production Excised roots were rinsed with distilled water thoroughly and then enwrapped gently with absorbent cotton which was wetted by treatment solution, placed immediately into a 10 cm3 glass jar for 1 h at room temperature. Preliminary experiments showed no correlation between wounding area and ethylene production, indicating that ethylene measured was not due to wounding. To investigate effect of ethylene synthesis inhibitors on ethylene production, 0.5 M aminoethoxyvinylglycin (AVG) and 10 M CoCl2 were used to treat M. falcata seedlings grown in +P and −P solutions for 48 h, and then ethylene production was measured as described above. Ethylene was sampled with 1 cm3 syringes from the headspace of the sealed container and the concentration was determined with a gas chromatograph (GC-7A, Shimadzu, Japan) fitted with a flame ionization detector and an activated glass column filled with GDX502 (Shimadzu, Japan). The detection limit was 0.013 cm3 m−3 . Ethylene production was calculated on the basis of fresh weight (FW) of root samples. Root fresh weight was determined immediately after the ethylene measurement. 2.3. Measurements of root hydraulic conductivity (Lpr) The steady-state flow rate (Jv) was measured using the hydrostatic pressure method as described by Wan and Zwiazek (1999). Briefly, a glass cylinder was inserted into a pressure chamber (PMS
Fig. 1. Effects of P deficiency on ethylene production from M. falcata roots in the absence and presence of 0.5 M aminoethoxyvinylglycin (AVG) (A) and 10 M CoCl2 (B). Seedlings of M. falcata grown in P-sufficient solution were transferred into P-deficient solution without or with 0.5 M AVG and 10 M CoCl2 for 48 h, and ethylene production from the excised roots was determined. Data are mean ± SD of four replicates. Different letters indicate significantly different values (P = 0.05).
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increased to 2.85 ± 1.92 ppm g−1 FW h−1 after 2-d of exposure of M. falcata seedlings to P-deficient solutions. Ethylene production from P-deficient roots was markedly inhibited by AVG and CoCl2 (Fig. 1B), the two chemicals that were used to inhibit activity of ACC synthase (ACS) and ACC oxidase (ACO), respectively (Lau and Yang, 1976). In contrast, AVG and CoCl2 did not have significant effect on Lpr of M. falcata seedlings grown in P-sufficient solutions (Fig. 1B). 3.2. P deficiency-induced reduction of root hydraulic conductivity (Lpr) To determine Lpr, we first calculated slope of water flow rates against the applied pressure for M. falcata seedlings grown in P-sufficient and P-deficient solutions. As shown in Fig. 2A, the slope for P-sufficient M. falcata seedlings was about two times greater than that for P-deficient seedlings. Given that Lpr is defined as the ratio of the slope to root surface area, the reductions in the slope induced by P deficiency led to a decrease in Lpr of M. falcata seedlings from 20.6 ± 2.9 to 14.6 ± 1.8 mg m−2 s−1 MPa−1 (n = 7) in response to transferring the seedlings from P-sufficient to P-deficient solutions for 48 h (Fig. 2B). Moreover, the P deficiencyinduced reduction in Lpr was rapidly recovered to the level similar to those for M. falcata seedlings grown in P sufficient solutions upon transferring the seedlings from P-deficient to P-sufficient solutions for 24 h (Fig. 2B).
Fig. 3. Effects of ethephone (A) and 1-aminocyclopropane-carboxylic acid (ACC) (B) on Lpr of M. falcata seedlings grown in P-sufficient (+P) and P-deficient (−P) solution. Lpr of M. falcata seedlings grown in P-sufficient and P-deficient solutions in the absence and presence of 5 M ethephone and 1 M ACC for 48 h was determined. Data are mean ± SD for at least seven seedlings for each treatment, and different letters indicate significantly different values (P = 0.05).
3.3. Ethylene is involved in P deficiency-induced reduction of Lpr
Fig. 2. Water flow rate vs the applied pressure for M. falcata seedlings grown continuously in P-sufficient (CK) and transferred into P-deficient solutions for 48 h (A). Data are mean ± SD of at least of five seedlings for each treatment. Root hydraulic conductivity of M. falcata seedlings grown in P-sufficient (CK), after transferred to P-deficient solution for 48 h (−P) and then transferred to the P-sufficient (+P) solutions for 24 h (B). Data are mean ± SD of seven seedlings for each treatment. Different letters indicate significantly different values (P = 0.05).
Because P deficiency evoked a marked ethylene production (Fig. 1) as well as induced a substantial reduction in Lpr (Fig. 2B), it is interesting to know whether the two events are related. To establish whether the P deficiency-induced ethylene production is associated with the P deficiency-induced inhibition of Lpr, we first studied effects of ethylene donor (ethephon) and ethylene biosynthesis precursor (ACC) on Lpr of M. falcata seedlings grown in P-sufficient and P-deficient solutions. As shown in Fig. 3A, treatment with ACC reduced Lpr of M. falcata seedlings grown in both P-sufficient and P-deficient solutions with Lpr of seedlings grown in P-sufficient solutions being more sensitive to ACC than that of seedlings grown in P-deficient solutions. For instance, 1 M ACC reduced Lpr of P-sufficient plants from 31.9 ± 2.1 to 11.1 ± 1.8 mg m−2 s−1 MPa−1 (n = 7), while the same treatment led to a reduction of Lpr of Pdeficient seedlings from 23.0 ± 1.9 to 16.7 ± 2.0 mg m−2 s−1 MPa−1 (n = 5). Treatment with 5 M ethephone (Eth) significantly reduced Lpr of P-sufficient seedlings, while it had no effect on Lpr of Pdeficient seedlings (Fig. 3B). These results imply that the reduction of Lpr under conditions of P deficiency may result from ethylene production. To test this possibility, we then evaluated the effect of inhibitors of ethylene biosynthesis (AVG, CoCl2 ) on Lpr of M. falcata seedlings grown in both P-sufficient and P-deficient solutions. AVG at 0.5 M had no effect on Lpr of P-sufficient seedlings (Fig. 4A). By contrast, Lpr values of P-deficient seedlings were increased significantly when the same concentration of AVG was used to treat M. falcata seedlings grown in P-deficient solutions (Fig. 4A). The Lpr values for P-deficient seedlings in the presence of AVG were comparable to those of P-sufficient seedlings in the absence of AVG
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Fig. 5. Effect of HgCl2 on Lpr of P-sufficient and P-deficient M. falcata seedlings. At time zero, 50 M HgCl2 was added into the incubation solution, and Lpr was determined after 10 min of exposure of the seedlings to HgCl2 was determined. Thereafter, 5 M dithiothreitol (DTT) was added to the incubation solution to washout HgCl2 , and Lpr was determined again in the presence of DTT. Data are mean ± SD for five seedlings for each treatment.
Fig. 4. Responses of Lpr of P-sufficient (+P) and P-deficient (−P) seedlings to 0.5 M aminoethoxyvinylglycine (AVG) (A) and 10 M CoCl2 (B). M. falcata seedlings were grown in the P-sufficient solution and then transferred into P-deficient solutions with and without AVG and CoCl2 for 48 h, and Lpr was then determined. Data are mean ± SD for at least seven seedlings for each treatment, and different letters indicate significantly different values (P = 0.05).
(Fig. 4A), suggesting that AVG can reverse the P deficiency-induced reduction in Lpr of M. falcata seedlings. A similar stimulatory effect of CoCl2 on Lpr of P-deficient seedlings, but not on Lpr of P-sufficient seedlings, was also found (Fig. 4B). These results indicate that ethylene production elicited by P deficiency is likely to account for the observed reduction of Lpr under P deficiency. 3.4. Lpr of P-sufficient seedlings is more sensitive to HgCl2 than that of P-deficient seedlings Mercury has been widely used as an effective antagonist of aqauporins in both animal and plant cells. To test whether the inhibition of aquaporin activities accounts for the P deficiency-induced decrease in Lpr, we studied effect of HgCl2 on Lpr of M. falcata seedlings grown in P-sufficient and P-deficient solutions. Treatment of M. falcata roots with 50 M HgCl2 rapidly reduced Lpr of P-sufficient seedlings from 27.2 ± 1.8 to 9.8 ± 3.4 mg m−2 s−1 MPa−1 (n = 7) (Fig. 5). When M. falcata seedlings grown in P-deficient solutions were exposed to the same concentrations of HgCl2 , Lpr was further reduced to an identical level to Lpr of P-sufficient seedlings treated with HgCl2 (Fig. 5). The reduction of Lpr for both P-sufficient and P-deficient seedlings was reversed by washing HgCl2 with reducing agent dithiothreitol (DTT) (Fig. 5). 4. Discussion A marked ethylene production from various plant species has been widely observed in response to nutrient deficiency in general (Lynch and Brown, 1997) and P deficiency in particular (Borch et al., 1999). On the other hand, a decrease in water flow across roots into root xylem due to reduction in root hydraulic conductivity has
also frequently reported in response to nutrient deficiency, including nitrate (Carvajal et al., 1996; Clarkson et al., 2000), phosphate (Radin and Eidenbock, 1984; Carvajal et al., 1996; Clarkson et al., 2000; Fan et al., 2007) and sulfate (Karmoker et al., 1991; Clarkson et al., 2000). In the present study, we demonstrated that exposure of M. falcata seedlings to P-deficient solutions for 2 d led to a significant decrease in Lpr (Fig. 2). More importantly, we found that treatment of M. falcata plants with ethylene donor ethephon and ethylene biosynthesis precursor ACC also reduced Lpr of M. falcata seedlings with P-sufficient seedlings being reduced greater than P-deficient plants (Fig. 3). These findings prompted us to speculate whether the P deficiency-induced ethylene production plays a role in P deficiency-induced reduction of Lpr. To test this possibility, we investigated effect of P deficiency on Lpr in the presence of ethylene synthesis antagonists of Co2+ and AVG, which markedly reduced ethylene production from M. falcata roots grown in Pdeficient solutions, but not those of grown in P-sufficient solutions (Fig. 1). We found that Co2+ and AVG had no effect on Lpr of Psufficient plants, but they enhanced Lpr of P-deficient seedlings to the level comparable to Lpr of P-sufficient seedlings (Fig. 4). These results suggest that the P deficiency-induced decrease in Lpr can be abolished by inhibition of ethylene production, thus highlighting an important role of ethylene in regulation of Lpr in P deficiency conditions. Because aquaporins are key regulators of water transport across roots (Javot and Maurel, 2002; Tyerman et al., 2002), we further examined effect of aquaporin antagonist, HgCl2 , on Lpr of P-sufficient and P-deficient seedlings. Our results showed that Lpr of P-deficient seedlings was less inhibited by HgCl2 than that of P-sufficient seedlings (Fig. 5), suggesting that inhibition of aquaporins may underlie the P deficiency-induced reduction of Lpr. Plant aquaporins are modulated by cytoplasmic pH and free Ca2+ activity ([Ca2+ ]c ) such that acidification of cytosol and elevation of [Ca2+ ]c inhibit aqauporins activity, leading to a decrease in osmotic permeability (Alleva et al., 2006). As ethylene can elicit a rapid increase in cytoplamsic [Ca2+ ]c by activating Ca2+ -permeable channels (Zhao et al., 2007), it is conceivable that P deficiency-induced ethylene production may inhibit aquaporin activity via affecting [Ca2+ ]c . In the present study, we observed a differential effect of AVG and Co2+ on ethylene production of M. falcata roots grown in P-deficient and P-sufficient solutions (Fig. 1). This finding may be accounted for by that the basal level of ethylene in M. falcata roots is relatively insensitive to AVG and CoCl2 due to low activities of both ACS and ACO under P-sufficient conditions. The greater inhibitory effect of AVG and CoCl2 on Lpr of P-deficient seedlings than that of P-sufficient seedlings indicates that the P deficiency enhances
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activities of ACS and ACO, thus leading to the marked ethylene production. The P deficiency-induced ethylene was abolished in the presence of AVG and CoCl2 by inhibiting ACS and ACO, respectively. A similar differential effect of AVG on ethylene production from chickpea nodules in the presence and absence of nitrate has been reported by Mann et al. (2002). Kamaluddin and Zwiazek (2002) reported that exogenous ethylene alleviates hypoxia-induced decrease in Lpr of aspen roots. In contrast to our results, Lpr of aspen plants in aerated control solutions was increased by 25% when ethylene gas was used to treat the plants (Kamaluddin and Zwiazek, 2002). Several possible explanations may account for the differences between our findings and those of Kamaluddin and Zwiazek (2002). These include differences in species used in the two studies, duration of ethylene treatment (2 d vs 12 h) and methods used for ethylene treatments (ACC and ethephon vs ethylene gas). Nevertheless, our findings and those of Kamaluddin and Zwiazek (2002) highlight the important role of ethylene played in regulation of water transport in plants by possibly modulating activity of aquaporins. It would be interesting to examine whether ethylene affects aqauporins at both transcriptional and protein levels in the future study. A number of studies have reported that P deficiency-induced a decrease in root Lpr of different plant species. These include wheat (Carvajal et al., 1996), maize (Fan et al., 2007) and cotton (Radin and Eidenbock, 1984). In wheat, Lpr was reduced by 75% after a 7-d P deprivation (Carvajal et al., 1996), while Fan et al. (2007) found that Lpr of maize roots was reduced by approx. 20% after 4-d removal of P from culture solution, and the P deficiency-induced reduction of Lpr increased with time such that Lpr was reduced by more than 50% after treatment with P-deprivation for 8 d. Unlike those studies in which phosphate was removed from incubating solutions (Radin and Eidenbock, 1984; Carvajal et al., 1996; Fan et al., 2007), we transferred M. falcata seedlings from P-sufficient solutions to P-deficient solutions containing 5 M phosphate. The P-deficient solutions used in our study may be more physiologically relevant than those used by others as a sudden reduction of phosphate availability to zero in soil would hardly occur. In addition, we focused on relatively short-term effect of P deficiency on Lpr as exposure of plants to P-deficient solutions for longer period would cause substantial changes in root morphology such as inhibition of primary root growth and stimulation of lateral root and root hair growth (Lynch and Brown, 2001), which will contribute to the changes in Lpr, particularly for those Lpr values expressed on the basis of root length (Radin and Eidenbock, 1984) and root weight (Carvajal et al., 1996). This point is reflected in the findings that the slope for flow rates against the applied pressure were reduced by 50% while the corresponding Lpr was only reduced by 27% in response to changing the phosphate concentrations from 500 to 5 M. The down-regulation of Lpr under nutrient deficiency has been accounted for by inhibition of aquaporins as suggested from experiments using aquaporin inhibitors of mercurial (Carvajal et al., 1996; Clarkson et al., 2000). A similar conclusion may also be drawn from the observation that less inhibition of Lpr of M. falcata seedlings grown in P-deficient solutions by HgCl2 than that of seedlings grown in P-sufficient solutions (Fig. 5). However, Lpr of P-deprived wheat plants is no longer sensitive to HgCl2 (Carvajal et al., 1996), while in our study we found that Lpr of P-deficient seedlings was further reduced to the level identical to that Lpr of P-sufficient plants (Fig. 5). These differences may be accounted for by that aquaporins in M. falcata roots is partly inhibited by the P-deficient treatment, while the prolonged exposure of wheat seedlings to P-deleted solution may completely inhibit aquaporins. Alternatively, the observed P deficiency-induced decrease in Lpr of M. falcata seedlings may result from formation of aerenchyma, which impedes water flow through the air space in roots, leading to lower Lpr (Fan et al., 2007). However, the formation of
aerenchyma in maize roots appears to occur after 4-d of exposure to P-deficient solutions (Fan et al., 2007), while in our study the decrease in Lpr was observed after 2-d exposure to P-deficient solutions. Further, the observation that P deficiency-induced reduction in Lpr was rapidly reversed upon exposure of the P-deficient seedlings to P-sufficient solutions (Fig. 2) may also argue against the contribution of aerenchyma formation to the reduced Lpr in our study. In conclusion, we demonstrate that P deficiency-induced reduction of root hydraulic conductivity in M. falcata seedlings is closely associated with ethylene production such that inhibition of ethylene production abolished the decrease in Lpr under P-deficient conditions. Recent study also reveals that ethylene can also induce stomatal closure (Desikan et al., 2006). Therefore, under P deficiency, ethylene may play an important role in regulation of plant water status via closing stomata and down-regulation of root hydraulic conductivity. This novel function of ethylene resembles that of ABA in terms of regulation of stomatal movement (Schroeder et al., 2001) and modulation of root hydraulic conductivity (Hose et al., 2000). Future research on the interactions between ABA and ethylene in control of root hydraulic conductivity is warranted. Acknowledgements This work was supported by the State Key Basic Research Development Program of China (2007CB106800) and Natural Science Foundation of China (No. 30821062 & 90817011) and research funding of the State Key Laboratory of Vegetation and Environmental Change (VEWALNE-project). References Alleva, K., Niemietz, C.M., et al., 2006. Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations. J. Exp. Bot. 57, 609–621. Barker, D.G., Pfaff, T., Moreau, D., Grove, E., Ruffel, S., Lepetit, M., Whitehand, S., Maillet, F., Nair, R.M., Journet, E.-P., 2006. Growing M. truncatula: choice of substrates and growth conditions. Medicago truncatula Handbook, http://www.noble.org/MedicagoHandbook/. Birner, T.P., Steudle, E., 1993. Effects of anaerobic conditions on water and solute relations, and on active-transport in roots of maize (Zea mays L). Planta 190, 474–483. Borch, K., Bouma, T.J., et al., 1999. Ethylene: a regulator of root architectural responses to soil phosphorus availability. Plant Cell Environ. 22, 425–431. Carvajal, M., Cooke, D.T., et al., 1996. Response of wheat plants to nutrient deprivation may involve the regulation of water-channel function. Planta 199, 372–381. Carvajal, M., Martinez, V., et al., 1999. Physiological function of water channels as affected by salinity in roots of paprika pepper. Physiol. Plant 105, 95–101. Clarkson, D.T., Carvajal, M., et al., 2000. Root hydraulic conductance: diurnal aquaporin expression and the effect of nutrient stress. J. Exp. Bot. 51, 61–70. Desikan, R., Last, K., et al., 2006. Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis. Plant J. 47, 907–916. Fan, M., Bai, R., et al., 2007. Aerenchyma formed under phosphorus deficiency contributes to the reduced root hydraulic conductivity in maize roots. J. Integra. Plant Biol. 49, 598–604. Hose, E., Steudle, E., et al., 2000. Abscisic acid and hydraulic conductivity of maize roots: a study using cell- and root pressure probes. Planta 211, 874–882. Javot, H., Maurel, C., 2002. The role of aquaporins in root water uptake. Ann. Bot. 90, 301–313. Kamaluddin, M., Zwiazek, J.J., 2002. Ethylene enhances water transport in hypoxic aspen. Plant Physiol. 128, 962–969. Karmoker, J.L., Clarkson, D.T., et al., 1991. Sulphate deprivation depresses the transport of nitrogen to the xylem and the hydraulic conductivity of barley (Hordeum vulgare L) roots. Planta 185, 269–278. Kimura, K., Yamasaki, S., 2003. Accurate root length and diameter measurement using NIH Image: use of Pythagorean distance for diameter estimation. Plant Soil 254, 305–315. Lau, O.-L., Yang, S.F., 1976. Inhibition of ethylene production by cobaltous ion. Plant Physiol. 58, 114–117. Lee, S.H., Chung, G.C., et al., 2005. Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and–tolerant figleaf gourd. J. Exp. Bot. 56, 985–995. Lopez-Berenguer, C., Garcia-Viguera, C., et al., 2006. Are root hydraulic conductivity responses to salinity controlled by aquaporins in broccoli plants? Plant Soil 279, 13–23. Lynch, J.P., Brown, K.M., 1997. Ethylene and plant responses to nutritional stresses. Physiol. Plant 100, 613–619.
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Phosphorus deficiency-induced reduction in root hydraulic conductivity in Medicago falcata is associated with ethylene production Yan-Su Li, Xiao-Tao Mao, Qiu-Ying Tian, Ling-Hao Li, Wen-Hao Zhang ∗ State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China
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Article history: Received 13 January 2009 Received in revised form 26 May 2009 Accepted 28 May 2009 Keywords: Phosphorus deficiency Root hydraulic conductivity Ethylene production Aquaporins Medicago falcata L
a b s t r a c t Plants grown in phosphorus-deficient solutions often exhibit disruption of water transport due to reduction in root hydraulic conductivity (Lpr) and enhanced ethylene production. To uncover the relationship between the reduction in Lpr and increase in ethylene production, we investigated effect of phosphorus (P) deficiency on ethylene production and Lpr in legume plants of Medicago falcata L. There was an increase in ethylene production and a reduction of Lpr of M. falcata roots when M. falcata seedlings grown in P sufficient solutions (0.5 mM H2 PO4 2− ) were transferred to P-deficient solutions (5 M H2 PO4 2− ). Antagonists of ethylene biosynthesis, CoCl2 and aminoethoxyvinylglycine (AVG), abolished the P deficiency-induced ethylene production. Root hydraulic conductivity of M. falcata seedlings grown in P-sufficient solutions was insensitive to CoCl2 and AVG, while the two chemicals enhanced Lpr for those grown in P-deficient solutions, suggesting that P deficiency-induced decrease in Lpr can be reversed by inhibiting ethylene production. Ethylene precursor 1-amino cyclopropane-1-carboxylic acid (ACC) and ethylene donor ethephon had greater inhibitory effect on Lpr of P-sufficient seedlings than that of P-deficient seedlings. Root hydraulic conductivity of P-sufficient seedlings was more sensitive to HgCl2 than that of P-deficient seedlings. Taken together, these findings suggest that ethylene induced by P deficiency may play an important role in modulation of root hydraulic conductivity by affecting aquaporins in plants. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Root hydraulic conductivity (Lpr), which mainly reflects the hydraulic resistance for radial water flow from rhizosphere to the root xylem, is a key parameter in regulation of plant water status by modulating water transport in roots (Steudle and Peterson, 1998). Root hydraulic conductivity has been shown to be sensitive to a number of environmental factors. These include hypoxia (Zhang and Tyerman, 1991; Birner and Steudle, 1993; Zhang and Tyerman, 1999), salinity (Carvajal et al., 1999; Lopez-Berenguer et al., 2006) and low temperature (Wan et al., 1999; Lee et al., 2005; Murai-Hatano et al., 2008). In plants, water flow across roots can occur along a combination of apoplastic, symplastic, and transcellular pathways (Steudle and Peterson, 1998). The dominant water transport pathways in roots are dependent on factors such as plant species, growth conditions, developmental stages and driving force for radial water flow across roots (Steudle and Peterson, 1998). The
Abbreviations: ACS, 1-aminocyclopropane-1-carboxylic acid synthease; ACO, 1aminocyclopropane-1-carboxylic acid oxidase; AVG, aminoethoxyvinylglycine; DTT, dithiothreitol; Eth, ethephone; Jv, steady-state flow rate; Lpr, root hydraulic conductivity. ∗ Corresponding author. Tel.: +86 10 6283 6697; fax: +86 10 6259 2430. E-mail address: [email protected] (W.-H. Zhang). 0098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2009.05.013
discovery of aquaporins as major proteins to facilitate water transport across membranes greatly advances our knowledge on plant water transport at both cellular and whole plant levels (Javot and Maurel, 2002; Tyerman et al., 2002). In this context, the changes in Lpr in response to abiotic stresses have frequently been ascribed to reductions in activities of aquaporins as demonstrated by antagonist of aquaporins and expression of genes encoding aqauporins (Martinez-Ballesta et al., 2003; Murai-Hatano et al., 2008). Like ion channels, recent studies have revealed that aquaporins can also be gated (opening and closing) by factors such as cytoplasmic pH and free Ca2+ activity (Tournaire-Roux et al., 2003; Alleva et al., 2006), mechanical stimuli (Wan et al., 2004) and phytohormones (Hose et al., 2000; Wan et al., 2004). Therefore, plants can respond to abiotic stress by regulating their Lpr via changes in aquaporin activity. There have been a number of reports demonstrating that Lpr is reduced in response to deficiency in anion nutrients such as phosphate (Radin and Eidenbock, 1984; Radin and Matthews, 1989; Carvajal et al., 1996; Clarkson et al., 2000; Fan et al., 2007) nitrate (Radin and Matthews, 1989; Carvajal et al., 1996; Clarkson et al., 2000) and sulfate (Karmoker et al., 1991; Clarkson et al., 2000) in different plant species. Root hydraulic conductivity is also sensitive to plant hormones. For instance, there are reports that Lpr is modulated by abscisic acid (ABA) (Hose et al., 2000; Wan et al., 2004) and ethylene (Kamaluddin and Zwiazek, 2002). In addition to changes in Lpr, P deficiency also induces ethylene production
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(Lynch and Brown, 1997; Borch et al., 1999; Schmidt, 2001). The P deficiency-induced ethylene production may be involved in modulation of root growth and development (Borch et al., 1999; Ma et al., 2003; Zhang et al., 2003). However, there has been no study to examine the relationship between P deficiency-induced ethylene production and reduction in Lpr under conditions of P deficiency. In the present study, we investigated effect of P deficiency on Lpr of legume plants of Medicago falcata, an important native legume plant widely distributed in Inner Mongolia grassland in northern China, and evaluated the role of ethylene in the changes in Lpr induced by P deficiency. 2. Materials and methods 2.1. Plant material Seeds of M. falcata L., which were obtained from the Institute of Grassland Research, the Chinese Academy of Agricultural Sciences, were dipped in sulfuric acid for approx. 5 min to destroy the seed capsule, and then rinsed thoroughly with sterilized water. Thereafter the seeds were spread on 0.75% agar to germinate at 25 ◦ C till the radicals being about 2 cm. The seedlings were transferred to plastic buckets filled with 5 L fully aerated nutrient solution (Barker et al., 2006). Each bucket contained 15 plants. Plants were grown in a growth chamber with 25 ◦ C (day)/20 ◦ C (night), 80% relative air humidity, 200–230 mol m−2 s−2 photosynthetic photon flux density and 16/8 h day–night period. After grown in the culture solution for 3 weeks, half of the plants were transferred to P-deficient (−P) solutions. Potassium phosphate was added to give a final concentration of 500 M of H2 PO4 − for P-sufficient (+P) and 5 M for P-deficient (−P) media, respectively. To maintain the identical concentrations of K between P-sufficient and P-deficient solutions, 455 M KCl was added in the P-deficient solutions. Other components of the hydroponic nutrient solution were: 1 mM MgSO4 , 0.25 mM K2 SO4 , 0.25 mM CaCl2 , 1 mM NH4 NO3 , 2.5 mM KNO3 , 100 M Fe–Na–EDTA, 30 M H3 BO3 , 5 M MnSO4 , 1 M ZnSO4 , 1 M CuSO4 , 0.7 M Na2 MoO4 and 50 M KCl. pH of the hydroponic solutions was adjusted to 6.0.
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Instruments, Corvallis, OR) and filled with the solution used for culturing plants. The de-topped root system of M. falcata seedlings grown in P-sufficient and P-deficient solutions in the absence or presence of various chemicals (AVG, CoCl2 , ACC and ethephone) was immediately sealed in the pressure chamber. An appropriate pressure was gradually applied with compressed air and maintained during the measurements. The exuded xylem sap from the detoped root system was continuously collected and weighed. Root flow rates of whole root systems were monitored for linearity for at least 30 min. The flow rate was plotted against the applied pressure between 0.2 and 0.8 MPa, and the resultant slope (K) of the curve was then used to calculate hydraulic conductivity after determining surface area (A) of roots by scanning the root system with a scanner and analyzing the root images by Image J software (Kimura and Yamasaki, 2003). Hydraulic conductivity of the whole root system (Lpr) was calculated by: Lpr = K/A. 3. Results 3.1. Exposure of M. falcata seedlings to P-deficient solution led to evolution of ethylene To test whether P deficiency affects ethylene production from roots of M. falcata, ethylene production in response to P deficiency was studied. As shown in Fig. 1A, a marked increase in ethylene evolution from M. falcata roots was observed when M. falcata seedlings were transferred from P sufficient (500 M) to P deficient (5 M) solutions for 2d. For instance, ethylene evolution from P sufficient roots was 0.20 ± 0.09 ppm g−1 FW h−1 , while this evolution was
2.2. Determination of ethylene production Excised roots were rinsed with distilled water thoroughly and then enwrapped gently with absorbent cotton which was wetted by treatment solution, placed immediately into a 10 cm3 glass jar for 1 h at room temperature. Preliminary experiments showed no correlation between wounding area and ethylene production, indicating that ethylene measured was not due to wounding. To investigate effect of ethylene synthesis inhibitors on ethylene production, 0.5 M aminoethoxyvinylglycin (AVG) and 10 M CoCl2 were used to treat M. falcata seedlings grown in +P and −P solutions for 48 h, and then ethylene production was measured as described above. Ethylene was sampled with 1 cm3 syringes from the headspace of the sealed container and the concentration was determined with a gas chromatograph (GC-7A, Shimadzu, Japan) fitted with a flame ionization detector and an activated glass column filled with GDX502 (Shimadzu, Japan). The detection limit was 0.013 cm3 m−3 . Ethylene production was calculated on the basis of fresh weight (FW) of root samples. Root fresh weight was determined immediately after the ethylene measurement. 2.3. Measurements of root hydraulic conductivity (Lpr) The steady-state flow rate (Jv) was measured using the hydrostatic pressure method as described by Wan and Zwiazek (1999). Briefly, a glass cylinder was inserted into a pressure chamber (PMS
Fig. 1. Effects of P deficiency on ethylene production from M. falcata roots in the absence and presence of 0.5 M aminoethoxyvinylglycin (AVG) (A) and 10 M CoCl2 (B). Seedlings of M. falcata grown in P-sufficient solution were transferred into P-deficient solution without or with 0.5 M AVG and 10 M CoCl2 for 48 h, and ethylene production from the excised roots was determined. Data are mean ± SD of four replicates. Different letters indicate significantly different values (P = 0.05).
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increased to 2.85 ± 1.92 ppm g−1 FW h−1 after 2-d of exposure of M. falcata seedlings to P-deficient solutions. Ethylene production from P-deficient roots was markedly inhibited by AVG and CoCl2 (Fig. 1B), the two chemicals that were used to inhibit activity of ACC synthase (ACS) and ACC oxidase (ACO), respectively (Lau and Yang, 1976). In contrast, AVG and CoCl2 did not have significant effect on Lpr of M. falcata seedlings grown in P-sufficient solutions (Fig. 1B). 3.2. P deficiency-induced reduction of root hydraulic conductivity (Lpr) To determine Lpr, we first calculated slope of water flow rates against the applied pressure for M. falcata seedlings grown in P-sufficient and P-deficient solutions. As shown in Fig. 2A, the slope for P-sufficient M. falcata seedlings was about two times greater than that for P-deficient seedlings. Given that Lpr is defined as the ratio of the slope to root surface area, the reductions in the slope induced by P deficiency led to a decrease in Lpr of M. falcata seedlings from 20.6 ± 2.9 to 14.6 ± 1.8 mg m−2 s−1 MPa−1 (n = 7) in response to transferring the seedlings from P-sufficient to P-deficient solutions for 48 h (Fig. 2B). Moreover, the P deficiencyinduced reduction in Lpr was rapidly recovered to the level similar to those for M. falcata seedlings grown in P sufficient solutions upon transferring the seedlings from P-deficient to P-sufficient solutions for 24 h (Fig. 2B).
Fig. 3. Effects of ethephone (A) and 1-aminocyclopropane-carboxylic acid (ACC) (B) on Lpr of M. falcata seedlings grown in P-sufficient (+P) and P-deficient (−P) solution. Lpr of M. falcata seedlings grown in P-sufficient and P-deficient solutions in the absence and presence of 5 M ethephone and 1 M ACC for 48 h was determined. Data are mean ± SD for at least seven seedlings for each treatment, and different letters indicate significantly different values (P = 0.05).
3.3. Ethylene is involved in P deficiency-induced reduction of Lpr
Fig. 2. Water flow rate vs the applied pressure for M. falcata seedlings grown continuously in P-sufficient (CK) and transferred into P-deficient solutions for 48 h (A). Data are mean ± SD of at least of five seedlings for each treatment. Root hydraulic conductivity of M. falcata seedlings grown in P-sufficient (CK), after transferred to P-deficient solution for 48 h (−P) and then transferred to the P-sufficient (+P) solutions for 24 h (B). Data are mean ± SD of seven seedlings for each treatment. Different letters indicate significantly different values (P = 0.05).
Because P deficiency evoked a marked ethylene production (Fig. 1) as well as induced a substantial reduction in Lpr (Fig. 2B), it is interesting to know whether the two events are related. To establish whether the P deficiency-induced ethylene production is associated with the P deficiency-induced inhibition of Lpr, we first studied effects of ethylene donor (ethephon) and ethylene biosynthesis precursor (ACC) on Lpr of M. falcata seedlings grown in P-sufficient and P-deficient solutions. As shown in Fig. 3A, treatment with ACC reduced Lpr of M. falcata seedlings grown in both P-sufficient and P-deficient solutions with Lpr of seedlings grown in P-sufficient solutions being more sensitive to ACC than that of seedlings grown in P-deficient solutions. For instance, 1 M ACC reduced Lpr of P-sufficient plants from 31.9 ± 2.1 to 11.1 ± 1.8 mg m−2 s−1 MPa−1 (n = 7), while the same treatment led to a reduction of Lpr of Pdeficient seedlings from 23.0 ± 1.9 to 16.7 ± 2.0 mg m−2 s−1 MPa−1 (n = 5). Treatment with 5 M ethephone (Eth) significantly reduced Lpr of P-sufficient seedlings, while it had no effect on Lpr of Pdeficient seedlings (Fig. 3B). These results imply that the reduction of Lpr under conditions of P deficiency may result from ethylene production. To test this possibility, we then evaluated the effect of inhibitors of ethylene biosynthesis (AVG, CoCl2 ) on Lpr of M. falcata seedlings grown in both P-sufficient and P-deficient solutions. AVG at 0.5 M had no effect on Lpr of P-sufficient seedlings (Fig. 4A). By contrast, Lpr values of P-deficient seedlings were increased significantly when the same concentration of AVG was used to treat M. falcata seedlings grown in P-deficient solutions (Fig. 4A). The Lpr values for P-deficient seedlings in the presence of AVG were comparable to those of P-sufficient seedlings in the absence of AVG
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Fig. 5. Effect of HgCl2 on Lpr of P-sufficient and P-deficient M. falcata seedlings. At time zero, 50 M HgCl2 was added into the incubation solution, and Lpr was determined after 10 min of exposure of the seedlings to HgCl2 was determined. Thereafter, 5 M dithiothreitol (DTT) was added to the incubation solution to washout HgCl2 , and Lpr was determined again in the presence of DTT. Data are mean ± SD for five seedlings for each treatment.
Fig. 4. Responses of Lpr of P-sufficient (+P) and P-deficient (−P) seedlings to 0.5 M aminoethoxyvinylglycine (AVG) (A) and 10 M CoCl2 (B). M. falcata seedlings were grown in the P-sufficient solution and then transferred into P-deficient solutions with and without AVG and CoCl2 for 48 h, and Lpr was then determined. Data are mean ± SD for at least seven seedlings for each treatment, and different letters indicate significantly different values (P = 0.05).
(Fig. 4A), suggesting that AVG can reverse the P deficiency-induced reduction in Lpr of M. falcata seedlings. A similar stimulatory effect of CoCl2 on Lpr of P-deficient seedlings, but not on Lpr of P-sufficient seedlings, was also found (Fig. 4B). These results indicate that ethylene production elicited by P deficiency is likely to account for the observed reduction of Lpr under P deficiency. 3.4. Lpr of P-sufficient seedlings is more sensitive to HgCl2 than that of P-deficient seedlings Mercury has been widely used as an effective antagonist of aqauporins in both animal and plant cells. To test whether the inhibition of aquaporin activities accounts for the P deficiency-induced decrease in Lpr, we studied effect of HgCl2 on Lpr of M. falcata seedlings grown in P-sufficient and P-deficient solutions. Treatment of M. falcata roots with 50 M HgCl2 rapidly reduced Lpr of P-sufficient seedlings from 27.2 ± 1.8 to 9.8 ± 3.4 mg m−2 s−1 MPa−1 (n = 7) (Fig. 5). When M. falcata seedlings grown in P-deficient solutions were exposed to the same concentrations of HgCl2 , Lpr was further reduced to an identical level to Lpr of P-sufficient seedlings treated with HgCl2 (Fig. 5). The reduction of Lpr for both P-sufficient and P-deficient seedlings was reversed by washing HgCl2 with reducing agent dithiothreitol (DTT) (Fig. 5). 4. Discussion A marked ethylene production from various plant species has been widely observed in response to nutrient deficiency in general (Lynch and Brown, 1997) and P deficiency in particular (Borch et al., 1999). On the other hand, a decrease in water flow across roots into root xylem due to reduction in root hydraulic conductivity has
also frequently reported in response to nutrient deficiency, including nitrate (Carvajal et al., 1996; Clarkson et al., 2000), phosphate (Radin and Eidenbock, 1984; Carvajal et al., 1996; Clarkson et al., 2000; Fan et al., 2007) and sulfate (Karmoker et al., 1991; Clarkson et al., 2000). In the present study, we demonstrated that exposure of M. falcata seedlings to P-deficient solutions for 2 d led to a significant decrease in Lpr (Fig. 2). More importantly, we found that treatment of M. falcata plants with ethylene donor ethephon and ethylene biosynthesis precursor ACC also reduced Lpr of M. falcata seedlings with P-sufficient seedlings being reduced greater than P-deficient plants (Fig. 3). These findings prompted us to speculate whether the P deficiency-induced ethylene production plays a role in P deficiency-induced reduction of Lpr. To test this possibility, we investigated effect of P deficiency on Lpr in the presence of ethylene synthesis antagonists of Co2+ and AVG, which markedly reduced ethylene production from M. falcata roots grown in Pdeficient solutions, but not those of grown in P-sufficient solutions (Fig. 1). We found that Co2+ and AVG had no effect on Lpr of Psufficient plants, but they enhanced Lpr of P-deficient seedlings to the level comparable to Lpr of P-sufficient seedlings (Fig. 4). These results suggest that the P deficiency-induced decrease in Lpr can be abolished by inhibition of ethylene production, thus highlighting an important role of ethylene in regulation of Lpr in P deficiency conditions. Because aquaporins are key regulators of water transport across roots (Javot and Maurel, 2002; Tyerman et al., 2002), we further examined effect of aquaporin antagonist, HgCl2 , on Lpr of P-sufficient and P-deficient seedlings. Our results showed that Lpr of P-deficient seedlings was less inhibited by HgCl2 than that of P-sufficient seedlings (Fig. 5), suggesting that inhibition of aquaporins may underlie the P deficiency-induced reduction of Lpr. Plant aquaporins are modulated by cytoplasmic pH and free Ca2+ activity ([Ca2+ ]c ) such that acidification of cytosol and elevation of [Ca2+ ]c inhibit aqauporins activity, leading to a decrease in osmotic permeability (Alleva et al., 2006). As ethylene can elicit a rapid increase in cytoplamsic [Ca2+ ]c by activating Ca2+ -permeable channels (Zhao et al., 2007), it is conceivable that P deficiency-induced ethylene production may inhibit aquaporin activity via affecting [Ca2+ ]c . In the present study, we observed a differential effect of AVG and Co2+ on ethylene production of M. falcata roots grown in P-deficient and P-sufficient solutions (Fig. 1). This finding may be accounted for by that the basal level of ethylene in M. falcata roots is relatively insensitive to AVG and CoCl2 due to low activities of both ACS and ACO under P-sufficient conditions. The greater inhibitory effect of AVG and CoCl2 on Lpr of P-deficient seedlings than that of P-sufficient seedlings indicates that the P deficiency enhances
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activities of ACS and ACO, thus leading to the marked ethylene production. The P deficiency-induced ethylene was abolished in the presence of AVG and CoCl2 by inhibiting ACS and ACO, respectively. A similar differential effect of AVG on ethylene production from chickpea nodules in the presence and absence of nitrate has been reported by Mann et al. (2002). Kamaluddin and Zwiazek (2002) reported that exogenous ethylene alleviates hypoxia-induced decrease in Lpr of aspen roots. In contrast to our results, Lpr of aspen plants in aerated control solutions was increased by 25% when ethylene gas was used to treat the plants (Kamaluddin and Zwiazek, 2002). Several possible explanations may account for the differences between our findings and those of Kamaluddin and Zwiazek (2002). These include differences in species used in the two studies, duration of ethylene treatment (2 d vs 12 h) and methods used for ethylene treatments (ACC and ethephon vs ethylene gas). Nevertheless, our findings and those of Kamaluddin and Zwiazek (2002) highlight the important role of ethylene played in regulation of water transport in plants by possibly modulating activity of aquaporins. It would be interesting to examine whether ethylene affects aqauporins at both transcriptional and protein levels in the future study. A number of studies have reported that P deficiency-induced a decrease in root Lpr of different plant species. These include wheat (Carvajal et al., 1996), maize (Fan et al., 2007) and cotton (Radin and Eidenbock, 1984). In wheat, Lpr was reduced by 75% after a 7-d P deprivation (Carvajal et al., 1996), while Fan et al. (2007) found that Lpr of maize roots was reduced by approx. 20% after 4-d removal of P from culture solution, and the P deficiency-induced reduction of Lpr increased with time such that Lpr was reduced by more than 50% after treatment with P-deprivation for 8 d. Unlike those studies in which phosphate was removed from incubating solutions (Radin and Eidenbock, 1984; Carvajal et al., 1996; Fan et al., 2007), we transferred M. falcata seedlings from P-sufficient solutions to P-deficient solutions containing 5 M phosphate. The P-deficient solutions used in our study may be more physiologically relevant than those used by others as a sudden reduction of phosphate availability to zero in soil would hardly occur. In addition, we focused on relatively short-term effect of P deficiency on Lpr as exposure of plants to P-deficient solutions for longer period would cause substantial changes in root morphology such as inhibition of primary root growth and stimulation of lateral root and root hair growth (Lynch and Brown, 2001), which will contribute to the changes in Lpr, particularly for those Lpr values expressed on the basis of root length (Radin and Eidenbock, 1984) and root weight (Carvajal et al., 1996). This point is reflected in the findings that the slope for flow rates against the applied pressure were reduced by 50% while the corresponding Lpr was only reduced by 27% in response to changing the phosphate concentrations from 500 to 5 M. The down-regulation of Lpr under nutrient deficiency has been accounted for by inhibition of aquaporins as suggested from experiments using aquaporin inhibitors of mercurial (Carvajal et al., 1996; Clarkson et al., 2000). A similar conclusion may also be drawn from the observation that less inhibition of Lpr of M. falcata seedlings grown in P-deficient solutions by HgCl2 than that of seedlings grown in P-sufficient solutions (Fig. 5). However, Lpr of P-deprived wheat plants is no longer sensitive to HgCl2 (Carvajal et al., 1996), while in our study we found that Lpr of P-deficient seedlings was further reduced to the level identical to that Lpr of P-sufficient plants (Fig. 5). These differences may be accounted for by that aquaporins in M. falcata roots is partly inhibited by the P-deficient treatment, while the prolonged exposure of wheat seedlings to P-deleted solution may completely inhibit aquaporins. Alternatively, the observed P deficiency-induced decrease in Lpr of M. falcata seedlings may result from formation of aerenchyma, which impedes water flow through the air space in roots, leading to lower Lpr (Fan et al., 2007). However, the formation of
aerenchyma in maize roots appears to occur after 4-d of exposure to P-deficient solutions (Fan et al., 2007), while in our study the decrease in Lpr was observed after 2-d exposure to P-deficient solutions. Further, the observation that P deficiency-induced reduction in Lpr was rapidly reversed upon exposure of the P-deficient seedlings to P-sufficient solutions (Fig. 2) may also argue against the contribution of aerenchyma formation to the reduced Lpr in our study. In conclusion, we demonstrate that P deficiency-induced reduction of root hydraulic conductivity in M. falcata seedlings is closely associated with ethylene production such that inhibition of ethylene production abolished the decrease in Lpr under P-deficient conditions. Recent study also reveals that ethylene can also induce stomatal closure (Desikan et al., 2006). Therefore, under P deficiency, ethylene may play an important role in regulation of plant water status via closing stomata and down-regulation of root hydraulic conductivity. This novel function of ethylene resembles that of ABA in terms of regulation of stomatal movement (Schroeder et al., 2001) and modulation of root hydraulic conductivity (Hose et al., 2000). Future research on the interactions between ABA and ethylene in control of root hydraulic conductivity is warranted. Acknowledgements This work was supported by the State Key Basic Research Development Program of China (2007CB106800) and Natural Science Foundation of China (No. 30821062 & 90817011) and research funding of the State Key Laboratory of Vegetation and Environmental Change (VEWALNE-project). References Alleva, K., Niemietz, C.M., et al., 2006. Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations. J. Exp. Bot. 57, 609–621. Barker, D.G., Pfaff, T., Moreau, D., Grove, E., Ruffel, S., Lepetit, M., Whitehand, S., Maillet, F., Nair, R.M., Journet, E.-P., 2006. Growing M. truncatula: choice of substrates and growth conditions. Medicago truncatula Handbook, http://www.noble.org/MedicagoHandbook/. Birner, T.P., Steudle, E., 1993. Effects of anaerobic conditions on water and solute relations, and on active-transport in roots of maize (Zea mays L). Planta 190, 474–483. Borch, K., Bouma, T.J., et al., 1999. Ethylene: a regulator of root architectural responses to soil phosphorus availability. Plant Cell Environ. 22, 425–431. Carvajal, M., Cooke, D.T., et al., 1996. Response of wheat plants to nutrient deprivation may involve the regulation of water-channel function. Planta 199, 372–381. Carvajal, M., Martinez, V., et al., 1999. Physiological function of water channels as affected by salinity in roots of paprika pepper. Physiol. Plant 105, 95–101. Clarkson, D.T., Carvajal, M., et al., 2000. Root hydraulic conductance: diurnal aquaporin expression and the effect of nutrient stress. J. Exp. Bot. 51, 61–70. Desikan, R., Last, K., et al., 2006. Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis. Plant J. 47, 907–916. Fan, M., Bai, R., et al., 2007. Aerenchyma formed under phosphorus deficiency contributes to the reduced root hydraulic conductivity in maize roots. J. Integra. Plant Biol. 49, 598–604. Hose, E., Steudle, E., et al., 2000. Abscisic acid and hydraulic conductivity of maize roots: a study using cell- and root pressure probes. Planta 211, 874–882. Javot, H., Maurel, C., 2002. The role of aquaporins in root water uptake. Ann. Bot. 90, 301–313. Kamaluddin, M., Zwiazek, J.J., 2002. Ethylene enhances water transport in hypoxic aspen. Plant Physiol. 128, 962–969. Karmoker, J.L., Clarkson, D.T., et al., 1991. Sulphate deprivation depresses the transport of nitrogen to the xylem and the hydraulic conductivity of barley (Hordeum vulgare L) roots. Planta 185, 269–278. Kimura, K., Yamasaki, S., 2003. Accurate root length and diameter measurement using NIH Image: use of Pythagorean distance for diameter estimation. Plant Soil 254, 305–315. Lau, O.-L., Yang, S.F., 1976. Inhibition of ethylene production by cobaltous ion. Plant Physiol. 58, 114–117. Lee, S.H., Chung, G.C., et al., 2005. Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and–tolerant figleaf gourd. J. Exp. Bot. 56, 985–995. Lopez-Berenguer, C., Garcia-Viguera, C., et al., 2006. Are root hydraulic conductivity responses to salinity controlled by aquaporins in broccoli plants? Plant Soil 279, 13–23. Lynch, J.P., Brown, K.M., 1997. Ethylene and plant responses to nutritional stresses. Physiol. Plant 100, 613–619.
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