Effects of drought and subsequent rewatering on

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Effects of drought and subsequent rewatering on Rumex obtusifolius leaves of different ages: reversible and irreversible damages a

a

Anna Katarina Gilgen & Urs Feller a

Institute of Plant Sciences, Section of Plant Nutrition, Oeschger Centre for Climate Change Research, University of Bern, Bern, Switzerland Published online: 12 Feb 2013.

Click for updates To cite this article: Anna Katarina Gilgen & Urs Feller (2014) Effects of drought and subsequent rewatering on Rumex obtusifolius leaves of different ages: reversible and irreversible damages, Journal of Plant Interactions, 9:1, 75-81, DOI: 10.1080/17429145.2013.765043 To link to this article: http://dx.doi.org/10.1080/17429145.2013.765043

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Journal of Plant Interactions, 2014 Vol. 9, No. 1, 7581, http://dx.doi.org/10.1080/17429145.2013.765043

RESEARCH ARTICLE Effects of drought and subsequent rewatering on Rumex obtusifolius leaves of different ages: reversible and irreversible damages Anna Katarina Gilgen* and Urs Feller Institute of Plant Sciences, Section of Plant Nutrition, Oeschger Centre for Climate Change Research, University of Bern, Bern, Switzerland

Downloaded by [54.152.109.166] at 11:05 07 September 2015

(Received 22 November 2012; accepted 7 January 2013) Solute leakage from leaves can be high, especially under abiotic stress. As climate models project higher drought risks for future summers in Central Europe, the more frequent and more severe drought stress periods might cause increased leakage. Solute leakage under drought and subsequent rewatering was tested in the weed Rumex obtusifolius. A strong increase in leakage from leaves was found in response to drought. Older leaves leaking high amounts of solutes after 17 days of drought were not able to recover and died. However, younger leaves leaking smaller amounts at the end of the drought period were able to recover during a subsequent rewatering period. The results show that the ability of leaves to recover from damage after a drought stress phase must be accounted for because it is an important factor for overall plant performance and finally for competition with other species in the field, particularly under drought conditions.

Keywords: water stress; nutrient loss; free amino acids; electrical conductivity; ion release

Introduction Plants can lose considerable amounts of solutes through leakage in the rain (Tukey 1966; Schenk & Feller 1990; Marschner 2012). Leakage is known to increase with leaf age (Pennazio et al. 1982; Shyr & Kao 1985; Debrunner & Feller 1995; Debrunner et al. 1999) and in response to both biotic and abiotic stresses, e.g. water shortage (Leopold et al. 1981; Vasquez-Tello et al. 1990; Takele 2010). Besides a loss of nutrients and under certain conditions also assimilates (Debrunner & Feller 1995; Debrunner et al. 1999), release of compounds affecting the growth of other species may be relevant as well (Carral et al. 1988; Zaller 2006). Climate change scenarios project decreasing precipitation and a rising heat wave frequency in Central Europe, leading to an increased risk for summer drought in the future (Frei et al. 2006; Christensen et al. 2007). Therefore, if summers will be drier and plants will be subjected to more frequent and/or more severe drought stress, this might influence the overall rate of leakage. So far, physiological investigations in this general context mainly focused on the stress period while the subsequent recovery phase was frequently neglected. Moreover, studies on the effects of drought stress on leakage were primarily performed using in vitro drought stress (Leopold et al. 1981; Stevanovic´ et al. 1997; Bajji et al. 2002) and only rarely assessed solute leakage from leaves of droughtstressed plants (Takele 2010; Filek et al. 2012). Rumex obtusifolius L. (broad-leaved dock), a very common weed, appears to be less susceptible to *Corresponding author. Email: [email protected] # 2013 Taylor & Francis

drought than co-occurring species in Central European grassland (Gilgen et al. 2010). In addition, a pot experiment under controlled conditions confirmed the high recovery potential of R. obtusifolius after drought (Gilgen & Feller 2013). Thus, the aim of this study was to assess drought resistance and consequently the competitiveness of the weed R. obtusifolius in grasslands under future climate conditions. Solute leakage from leaves of R. obtusifolius was measured both after a drought period and a succeeding longer recovery phase. This information serves as an indicator for drought effects on membranes as previously suggested by Bajji et al. (2002). It is to be expected that the accessibility of solutes for leakage in the rain increases in leaves after drought. The potential of individual leaves to recover should be addressed after rewatering and leaf age needs to be considered as well.

Materials and methods R. obtusifolius seeds originating from the region of Bern (Switzerland) were germinated on coarse quartz sand with deionized water. After 16 days, seedlings were supplied with a standard nutrient solution (according to Page et al. 2012). Twenty-four plants were transferred into 0.8 L pots (one plant per pot) at the age of approximately one month (44 days after putting seeds on quartz sand). The pots contained a nutrient-rich soil mixture (see Gilgen & Feller 2013). On average, plants had grown four to five leaves at the time of potting. Pots were randomly positioned


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on two shelves of a climate cabinet, and positions were rotated every 10 days (random procedure). Light in the climate cabinet was supplied with 55 W lamps at an intensity of around 120130 mmol m 2 s 1 on pot level. The cabinet was set to a cycle of 14 h light at 248C and 10 h darkness at 168C. Twelve of the 24 pots were randomly assigned to the control treatment and the other half to the drought treatment. Eight of the pots in each treatment were equipped with soil water potential sensors (Watermark Sensor, Irrometer Company, Inc., Riverside, CA, USA). During the first three weeks on soil, all pots were regularly irrigated to keep them well saturated. Afterward, the 12 pots assigned to the drought treatment did not receive water for 17 days. Regular irrigation of control pots was maintained as before. After these 17 days of treatment, six pots from the control and six pots from the drought treatment were harvested (see below), while all other pots were regularly irrigated from this point onward. Another 11 days later, the remaining plants from both treatments (six each) were harvested. The use of water was monitored by weighing the pots before and after each irrigation event. In order to compare evapotranspiration, drought pots were also weighed throughout the drought treatment whenever control pots were irrigated. Since plants close their stomata in response to drought and prevent excessive water loss (Chaves 1991; Signarbieux & Feller 2012), transpiration is decreased and, in parallel, leaf temperature increases. Therefore, leaf surface temperature can be used as an indicator for transpiration and stomatal opening, thereby indicating the level of stress in various plant parts (Jones et al. 2009; Costa et al. 2012). For a high time resolution of the developing drought stress, infrared pictures were taken using a thermal camera (TiR1, Fluke Corporation, Everett, WA, USA). At the two harvests, a leaf segment (2 cm 2 cm) was cut from each leaf. Leaves that had already been shed from the plant were not sampled and the still rolled youngest leaves were not analyzed as no welldefined area could be sampled. The leaf surface was quickly rinsed with deionized water before sampling to remove dust. Leaf segments were then soaked in 8 mL deionized water at 48C for 2 h in the dark. The collected leakage solutions were stored at 48C until analysis. The concentration of free amino acids was determined using the ninhydrin reaction as described by Debrunner and Feller (1995). Depending on the free amino acid content of the samples, either 250 mL or 100 mL of the leakage solution was used. Absorption at 578 nm was used to calculate free amino acid contents with known contents of L-alanine as a reference. Electrical conductivity (a measure for total ion concentration) was determined with a conductivity meter (CDH-42, OMEGA Engineering, Inc., Stam-

ford, CT, USA) after mixing 100 mL leakage solution with 2 mL deionized water. Contents of potassium (K) and magnesium (Mg) were determined by atomic absorption spectrometry (SpectrAA 220FS, Varian Techtron, Mulgrave, Australia). The leakage solution was adequately diluted using 1000 ppm Cs as CsCl in 0.1 N HCl for K and 5000 ppm La as LaCl3 in 0.1 N HCl for Mg. For statistical analyses, leaves were grouped according to their position at the beginning of the treatment (i.e. the last irrigation event for drought plants). The youngest leaf at this time was thus assigned position 0. All older leaves got negative numbers while younger leaves were denoted with positive numbers. Results are expressed on a leaf area basis. As most of the data were not normally distributed (tested with a ShapiroWilk test), nonparametric Wilcoxon rank-sum tests were used to compare the two treatments. All analyses were performed by using R software (version 2.14.2, R Development Core Team 2012). Results The drought treatment resulted in a steep and gradual decrease of soil water potential and daily evapotranspiration (Figure 1a,b). The difference to control conditions was significant six and five days after cessation of irrigation, respectively. After 17 days of drought, soil water potential was as low as 96.33 kPa (97.33 kPa) compared to only 1.60 kPa (90.40 kPa) in control pots. Given the type of sensors and the size of the pots, the absolute readings of soil water potential should be considered with care. However, the progressive drop of the soil water potential during the drought period is clearly documented. Daily evapotranspiration had dropped to less than 5 g H2O day 1 at the end of the drought treatment, while water consumption in control plants was almost 30 times higher. Plant growth stopped during a later phase of the drought treatment (i.e. constant number of leaves; Figure 1c). Soil water potential was immediately restored by rewatering. In contrast, daily evapotranspiration recovered only slowly and did not reach control levels after 11 days of rewatering (Figure 1b). Although a certain memory effect of the previous drought treatment in photosynthetically active leaves cannot be ruled out, the daily evapotranspiration was lowered by the still significantly reduced number of leaves compared to control plants (Figure 1c). The four parameters analyzed to quantify leakage (free amino acids, electrical conductivity, K, and Mg) displayed similar patterns. After 17 days of drought, high leakage from all leaves, except from the youngest, was observed in drought-stressed plants while only the oldest leaves showed increased leakage in control plants (Figures 25). After 11 days of rewatering, control plants still only leaked from old (i.e. senescing) leaves, while leaves of plants that had


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Figure 2. Leakage of free amino acids from R. obtusifolius leaves. Leaf 0 denotes the youngest expanded leaf at the beginning of the drought treatment. Leaves assigned a negative number are thus older than this leaf while leaves with positive numbers emerged during the treatment and are thus younger. Averages and standard errors are shown (n 6). Significant treatment differences according to Wilcoxon rank-sum tests are given as *0.05 ] P 0.01, **0.01 ] P 0.001. $ denotes dead leaves.

Figure 1. Soil water potential (a), daily evapotranspiration (b), and number of leaves (c) of R. obtusifolius during a drought period of 17 days and the subsequent recovery phase. Averages and standard errors are shown [n: until end of drought treatment (day 17): (a) control: 8, drought: 7, (b) and (c) 12; after end of drought treatment: (a) control: 3, drought 4, (b) and (c) 6].

previously been drought stressed were able to recover. Drought-stressed plants had lost most of their leaves during the previous 17 days of drought, and the oldest remaining leaf (which happened to be the youngest at the start of the treatment) showed very high leakage after 11 days of rewatering. However, the next younger leaves showed only minor leakage. This is remarkable since leaf 1 and leaf 2 (to a somewhat lesser extent) had shown increased leakage after 17 days of drought. The leakage of free amino acids in control plants was negligible throughout the experiment (Figure 2). Only the two oldest leaves showed minor amounts of leakage (B0.5 mmol cm 2). In contrast, considerable amounts of free amino acids (two to three times the amount of senescing control leaves) leaked from all leaves of drought-stressed plants. Even the youngest leaf leaked amounts comparable to those from the

senescing oldest leaf of control plants. However, young leaves developing during the stress were able to recover during rewatering and did not leak higher amounts of free amino acids than those of control plants at the end of the experiment (Figure 2b). The response of electrical conductivity (as a measure for total ion leakage) to the treatment was comparable to that of free amino acids. Very low electrical conductivity was measured in leakage solution from control plants. Again, the two oldest leaves showed slightly increased leakage (Figure 3). Electrical conductivity was significantly higher in leakage solution from drought-stressed plants. Only the two leaves that had developed during the 17 days of drought were able to keep leakage at a level lower than that of senescing control leaves. Also electrical conductivity of leakage solution from younger leaves was able to recover during rewatering. The pattern observed for electrical conductivity was reflected in leakage of K and Mg (Figures 4 and 5). The absolute contents of K in the leakage solution were up to 50 times higher than contents of Mg but high contents of the two ions co-occurred in the same leaves. Thermal (i.e. infrared) pictures suggested a gradual recovery of the leaves after drought stress (Figure 6). The first leaf to recover and displaying leaf temperatures comparable to those of control plants


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Figure 3. Electrical conductivity in the leakage solution from R. obtusifolius leaves. Leaf 0 denotes the youngest expanded leaf at the beginning of the drought treatment. Leaves assigned a negative number are thus older than this leaf while leaves with positive numbers emerged during the treatment and are thus younger. Averages and standard errors are shown (n 6). Significant treatment differences according to Wilcoxon rank-sum tests are given as *0.05 ] P 0.01, **0.01 ] P 0.001. $ denotes dead leaves.

Figure 4. Leakage of potassium (K) from R. obtusifolius leaves. Leaf 0 denotes the youngest expanded leaf at the beginning of the drought treatment. Leaves assigned a negative number are thus older than this leaf, while leaves with positive numbers emerged during the treatment and are thus younger. Averages and standard errors are shown (n 6). Significant treatment differences according to Wilcoxon rank-sum tests are given as *0.05 ] P 0.01, **0.01 ] P 0.001. $ denotes dead leaves.

was the youngest leaf. The next older leaf took some more days to recover but seemed to be able to do so as well. After 11 days of rewatering, newly developed leaves displayed average leaf cooling and older leaves, even though they started to senesce, were no longer heated up as they had been during the drought phase.

membrane functioning and allows solutes to leak from leaf surfaces (Simon 1974). Membrane functions would then be restored under rewatering and leakage would cease. This theory is consistent with our findings. In particular, the leakage of large amounts of free amino acids from drought-stressed leaves is an indication for damage to certain molecular structures. Under control conditions, no leakage of free amino acids was found in wheat, not even from senescent leaves (Debrunner & Feller 1995; Debrunner et al. 1999). Thus, if free amino acids leak, this might be caused by disturbances in the metabolism (e.g. proteolysis), in the long-distance translocation via the phloem and in membrane intactness. Both K and Mg have previously been shown to leak quite easily (Tukey 1970; Debrunner & Feller 1995). Although the drought response of free amino acid and ion leakage was similar, the shape of the response curves differed remarkably. While similar amounts of K and Mg leaked from all old leaves, free amino acid leakage showed a bell-shaped drought response, i.e. it was smaller in oldest and youngest leaves and highest at intermediate leaf age. In contrast to K and Mg, amino acids are metabolized in plants. Results indicate that a part of the free amino acids was removed from the oldest leaves. If

Discussion The damage caused by a drought period of 17 days was irreversible in older leaves of R. obtusifolius, and those leaves died after the drought period. Leakage from the oldest remaining leaf increased until the end of the rewatering phase but transpiration was restored and leaf temperatures consequently decreased to control values indicating that the damage was partially reversible. The increased leakage from older leaves is in accordance with previous findings (Leopold et al. 1981; Debrunner & Feller 1995). The younger leaves (i.e. leaves developed during the stress or later) were fully recovered after 11 days of rewatering. Mechanisms for leakage are still unclear (Marschner 2012). Although it was recently found that electrolyte leakage during senescence is not necessarily a symptom of damage to the cell membrane (Rolny et al. 2011), leakage is generally used as an estimate for cell membrane intactness (e.g. Bajji et al. 2002). Drought stress thus disturbs the



Figure 5. Leakage of magnesium (Mg) from R. obtusifolius leaves. Leaf 0 denotes the youngest expanded leaf at the beginning of the drought treatment. Leaves assigned a negative number are thus older than this leaf while leaves with positive numbers emerged during the treatment and are thus younger. Averages and standard errors are shown (n 6). Significant treatment differences according to Wilcoxon rank-sum tests are given as *0.05 ] P 0.01, **0.01 ] P 0.001. $ denotes dead leaves.

free amino acids were transported to younger leaves, this transport must have been more efficient than for K and Mg as these two elements are known to be mobile in the phloem (Marschner 2012). In theory, they should thus be redistributed from senescing

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leaves to younger leaves. However, the findings of this study suggest that this did not happen under drought stress, probably because the overall sink capacity was decreased (e.g. less new leaves produced, negative effects on the expansion of young leaves). It is remarkable that all plants were able to maintain the youngest leaf at the beginning of the stress but not any of the older leaves. It seems that R. obtusifolius managed to keep the youngest leaves alive by shedding all older leaves. This makes it a good competitor under drought stress. Solute leakage has been used as an indicator for drought resistance in grasses (Bajji et al. 2002; Filek et al. 2012) and legumes (Vasquez-Tello et al. 1990). The present study suggests that it could also be a possible diagnostic tool in nonleguminous forbs. However, the results indicate that the focus should not be on leakage at the end of the drought period but on the potential of leaves to recover during rewatering after drought. High solute losses due to drought, as observed for R. obtusifolius, do not necessarily mean that a plant cannot survive the drought. Actually, a decrease in solute leakage under rewatering is a good indicator for drought resistance of a species. Nevertheless, it needs to be kept in mind that the experimental plants were protected from water and no solutes were lost during the treatment. Under field conditions, R. obtusifolius plants might still lose a considerable amount of solutes due to leakage, although they are able to repair the damage in their structure. Depending on the nature of substances leaking during drought, the competition of R. obtusifolius with other species could be influenced. It has been reported by several groups that substances

Figure 6. Photographs and thermal pictures of a control and a drought-stressed R. obtusifolius plant at three points in time during the experiment. m labels the youngest leaf at the beginning of the treatment (leaf 0) and ' the first leaf built during the treatment (leaf 1).


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present in the leaves of R. obtusifolius are able to suppress neighboring species (Carral et al. 1988; Zaller 2006). In this context, it must be also borne in mind that the abundance of R. obtusifolius is known to be correlated with soil K concentration (Humphreys et al. 1999; Hann et al. 2012). Large amounts of K may leak in the rain from leaves of this species and may locally increase the K level in the soil around the R. obtusifolius plants. The behavior of this weed species during stress and subsequent recovery phases must be considered as an important aspect for its growth and distribution in a changing climate. This study led to the following main conclusions: (1) solute leakage can be used as a powerful technique to analyze damages in leaves during a drought period as well as during the subsequent recovery phase; (2) infrared pictures allow a high spatial and temporal resolution of reversible and irreversible droughtinduced damages; (3) an anticipated senescence of the older leaves of R. obtusifolius occurred during the stress and the recovery phase; (4) the fact that young leaves recovered well from drought stress must be considered as an important aspect for the performance of this weed species and the competition with other species in grasslands. References Bajji M, Kinet J, Lutts S. 2002. The use of the electrolyte leakage method for assessing cell membrane stability as a water stress tolerance test in durum wheat. Plant Growth Regul. 36:6170. Carral E, Reigosa MJ, Carballeira A. 1988. Rumex obtusifolius L: release of allelochemical agents and their influence on small-scale spatial distribution of meadow species. J Chem Ecol. 14:17631773. Chaves MM. 1991. Effects of water deficits on carbon assimilation. J Exp Bot. 42:116. Christensen JH, Hewitson B, Busuioc A, Chen A, Gao X, Held I, Jones R, Kolli RK, Kwon W-T, Laprise R, et al. 2007. Regional climate projections. In: Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, Tignor M, Miller HL, editors. Climate change 2007: the physical science basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge: Cambridge University Press; p. 847940. Costa JM, Ortunõ MF, Lopes CM, Chaves MM. 2012. Grapevine varieties exhibiting differences in stomatal response to water deficit. Funct Plant Biol. 39:179189. Debrunner N, Feller U. 1995. Solute leakage from detached plant parts in winter wheat: influence of maturation stage and incubation temperature. J Plant Physiol. 145:257260. Debrunner N, Von Lerber F, Feller U. 1999. Solute losses from various shoot parts of field-grown wheat by leakage in the rain. In: Anaç D, Martin-Pre´vel P, editors. Improved crop quality by nutrient management. Dordrecht: Kluwer Academic Publishers; p. 131134.

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