Spatial and temporal effects of nitrogen addition on. root life span of Leymus chinensis in a typical steppe of Inner Mongolia

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1 Functional Ecology 2008, 22, doi: /j x Spatial and temporal effects of nitrogen addition on Blackwell Publishing Ltd root life span of Leymus chinensis in a typical steppe of Inner Mongolia W.-M. Bai, Z.-W. Wang, Q.-S. Chen, W.-H. Zhang and L.-H. Li* Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing , China Summary 1. To understand root dynamics of the native, perennial clonal grass Leymus chinensis in Inner Mongolia steppe, we examined spatial and temporal effects of N addition on root survivorship and longevity traits of L. chinensis by studying responses of survivorship of roots distributed in three soil layers (0 10, and cm) and born in three seasons (spring, summer and autumn) to N addition with modified rhizotron technique during the growing seasons of Kaplan Meier analysis was used to generate survival functions and to estimate mean root life spans. 2. Roots in the three soil layers displayed similar life spans with mean overall value of 81 days in the control plots. N addition reduced life span of roots in the soil layers of 0 10 cm and cm, but it had no effect on life span of roots in the soil layer of cm. 3. Roots born in the three seasons differed in their life spans with the life span values of 21 58, and days for those roots born in spring, summer and autumn, respectively. N addition markedly reduced life span of roots born in summer in the three soil layers with the greatest reduction occurring in roots present in soil layer of cm, but it had marginal effect on life span of the spring-born and autumn-born roots. 4. The differential sensitivity of survivorship and longevity of L. chinensis roots occurred in different soil depths and born in different seasons to N addition highlights the importance of spatial and temporal characteristics of roots in response to soil N availability in Inner Mongolia steppe. Key-words: Leymus chinensis, N addition, root longevity, root survival, soil layers, spatial and temporal effect Introduction Life span of roots, especially of fine roots, have substantial effects on their ability to acquire and conserve soil resources for plants (Gill & Jackson 2000; Watson et al. 2000; López, Sabaté & Gracia 2001). However, root life span is a highly variable parameter, ranging from a few weeks to a number of years (Eissenstat & Yanai 1997), and is closely dependent on the size, species and mycorrohizal status of roots (Hendricks, Nadelhoffer & Aber 1993; Eissenstat & Yanai 1997; West, Espeleta & Donovan 2003). Recent studies have revealed that root longevity is sensitive to many biotic and abiotic factors (Cote et al. 1998; Gill et al. 2002; Anderson et al. 2003). A better understanding of root longevity and its regulatory mechanism is of critical importance in elucidating the strategies *Correspondence author. llinghao@ibcas.ac.cn of plants in their adaptation to unfavourable habitats and of nutrient use efficiency (Eissenstat & Yanai 1997). As the most limiting nutrient to plant growth and net primary productivity in semi-arid terrestrial ecosystems, soil nitrogen (N) availability is an important factor controlling root growth and life span (Pregitzer et al. 1998; Burton, Pregitzer & Hendrick 2000). There have been reports indicating that root life span is related to soil N status (Vogt, Grier & Vogt 1986; Pregitzer, Hendrick & Fogel 1993; Ryser 1996; Van der Krift & Berendse 2002). However, most studies investigating dependence of root longevity on soil N availability have been conducted by comparing root longevity of plants grown in nutrient-rich and -poor habitats (Burton et al. 2000; Phillips et al. 2006). As fertile and infertile habitats may not differ in N availability exclusively, differences in availability of other nutrients in addition to N as well as differences in soil physical characteristics may also exist. These differences would account for the contrasting results reported in the 2008 The Authors. Journal compilation 2008 British Ecological Society

2 584 W.-M. Bai et al. literature on correlations between soil N availability and root longevity. Therefore, more detailed studies are warranted to examine effect of N addition on root longevity of plants in general and herbaceous species in particular. In perennial woody species, root life span is dependent upon seasonality and soil depth (López et al. 2001; Anderson et al. 2003; Baddeley & Waston 2005). It remains unknown whether similar characteristics are also present in perennial herbaceous species. Moreover, although there have been a number of studies focusing on correlations between soil N status and root longevity (Burton et al. 2000; Van der Krift & Berendse 2002), no study has been conducted to investigate responses of survivorship and longevity of roots present in different soil depths and born in different seasons to soil N availability in herbaceous species. In addition to N, C supply from shoot to root is also associated with root longevity (Farrar & Jones 2000; Norby & Jackson 2000). A decrease in C allocation to root has been suggested to reduce root life span and/or root production (Eissenstat & Yanai 1997). The observation that reduced C input into roots increased root mortality of European Pinus sylvestris (Hogberg et al. 2001) is consistent with this proposition. Further, longevity of fine roots in Fraxinus mandshurica was positively correlated with content of soluble carbohydrate in the fine roots (Xu et al. 2006). These observations are indicative of reduced carbon allocation being likely to be an important mechanism to control life span of fine roots in trees. However, whether a similar mechanism also exists in herbaceous species remains to be deciphered. Leymus chinensis, a dominant clonal species in temperate Eurasian grasslands, is an important grass for grazing in Inner Mongolia steppe. But little is known about the species in terms of its biological and ecological characteristics. The overall aim of this study is to characterize responses of root survivorship and longevity traits of L. chinensis in a typical steppe community to N addition through a 2-year field study. More specifically, we investigated temporal and spatial characteristics of root survivorship and life span by studying effects of N addition on survivorship and life span of roots distributed in different soil layers and born in different seasons. Methods STUDY SITE The experiment was conducted in a temperate steppe in Duolun County, Inner Mongolia (41 46 N, E, altitude 1215 m), China. The soil type is chestnut soil with low N content and soil water holding capacity. Soil bulk density (0 30 cm soil layer) ranges from 1 43 to 1 67 g cm 3, mean soil ph value is 6 77 and soil texture is sandy. Climate is characterized by a semi-arid continental monsoon precipitation pattern, with a mean annual air temperature of 2 1 C. The maximum mean monthly temperature is about 18 7 C occurring in July, and the minimum mean temperature is 18 3 C occurring in January. Mean annual precipitation is about 386 mm, while mean potential evaporation is mm. The plant community at the experimental site is dominated by L. chinensis which accounted for over 95% of the total above-ground biomass, associated with sparsely distributed Hierrochloe glabra and Polygonum divaricatum (Wang et al. 2004). Plants start to growth in mid of April and senesce in the late September in this area. EXPERIMENTAL DESIGN In early September 2003, an area of 15 ha was fenced to exclude livestock grazing. Ten 5 8 m plots were arranged into two rows and five columns with a 1-m buffer distance between any two plots. The two plots in each column were randomly assigned to the following treatments: Control and N addition with five replicates for each treatment. N in the form of 32 g m 2 urea was added directly to the soil surface on 11 September INSTALLATION OF GLASS WINDOWS On 11 September 2003, one glass-root window was installed in each plot. The glass window (0 4 cm thick) of 40 cm in height and 50 cm in length was installed vertically into the soil. On each glass window, a cm panel (with 10 cm distance to the bottom and 5 cm to the right and left sides) was separated into twelve cm squares by carving the glass. In order to minimize the impacts of light on root growth, the upper edge of the glass window was installed under the soil surface and a piece of dark iron (50 cm in length, 1 5 cm in breadth and 0 5 mm in thickness) was covered on the top of the glass. In order to install the glass window, we dug a hole in each plot with a vertical profile with shovel. The glass window was put tightly to the profile and fixed with one iron stick in each side. After root windows were inserted, soil was backfilled as tightly. The soil was closely attached to the glass throughout the whole study periods. IMAGE COLLECTION A Canon G5 digital camera was used to observe the growth of roots. Observations began on 11 May 2004, which was about 7 months after the installation of glass windows, and lasted till 12 September 2005 with sampling intervals of approximately 10 days. On each observation, the soil at one side of the glass window was removed and cleaned with tissue paper. One digital picture was taken for each of the twelve cm numbered squares. Removed soil was backfilled after pictures were taken. IMAGE PROCESSING The software of Mapinfo professional (5 0) and the mouse of PC were employed to trace the process of appearance and disappearance of fine-roots. For the first collected images, each root was assigned with an identification number and classified as living or dead root based upon colour, and the same criteria were used throughout this study. For determining root production, root length for each individual root was simultaneously measured with an accuracy of 0 01 mm. The criteria for distinguishing dead from living roots were similar to those used by West et al. (2003), that is, those roots display symptoms with black colour and decaying characteristics of softening, shrivelling and/or partial decomposition are assumed to be dead. For subsequent image sets, the tracings from the previous date were overlaid on the new image, allowing previously existing roots to be identified. Newly emerged roots were also identified and numbered. Roots that disappeared and judged to be dead using the above criteria at the subsequent images were used to determine life span.

3 Root lifespan of Leymus chinensis and N addition 585 ANALYSIS OF ROOT SURVIVAL Root life span was calculated as the date on which roots were observed as black or disappeared minus the date on which the roots were initially observed on the window. The date of root appearance or disappearance was estimated as the date midway of the sampling period because they might have occurred on any day during the approximately 10-day sampling interval between two consecutive observations (Hooker et al. 2000; López et al. 2001; Anderson et al. 2003). We selected total of 2641 new roots germinated in spring (11 23 May), summer (19 30 July) and autumn (24 August September 4) of 2004 to analyse the effects of root birth timing and N addition on their survival rates and longevity. We calculated their mean longevity through a survival curve using Kaplan Meier method with SPSS (12 0) software, and compared the root survival rates by Log-rank test. Leymus chinensis roots are dominated by fine, adventitious roots with diameter < 0 5 mm. A stratified Cox proportional hazards regression was used to estimate the effects of treatment, soil depth and birth season on root life span as described by Gill et al. (2002). In the Cox model, the hazard for an individual root at time t is computed based upon the combination of a nonparametric baseline (h 0 ) function and an exponential function of k covariates: h i (t) = h 0 (t)exp(β 1 χ i β k χ ik ) The Cox model was used to estimate β coefficient for each model covariate and test the null hypothesis of β = 0 with a χ 2 statistic. A negative β coefficient indicates a decreased risk of mortality with an increase in the covariate values. A risk ratio (exp β) being used to calculate the percent change in the risk of mortality with a one unit change in the covariate was calculated as well (Wells & Eissenstat 2001; Gill et al. 2002). ROOT PRODUCTION AND MORTALITY Protocols used for root production and mortality in the present study mainly followed those of Burton et al. (2000). Briefly, root production for each sampling period was measured by summing the length of all new roots and adding the extension growth of all previously existing roots. Root mortality was determined by summing the lengths of all roots that had died during that interval and adding the length of existing roots disappeared due to herbivore or dieback. Production and mortality were both expressed as root length per glass rhizotron area observed (mm cm 2 ). MEASUREMENTS OF SOIL MOISTURE AND NITROGEN Soil water content at the depths of 0 10, and cm was measured gravimetrically on the same day root images were taken. Repeated measure ANOVA was used to analyse the difference in soil water content between treatments and between soil layers. In the following year (2004) of treatments, soil samples at the depths of 0 10, and cm were taken in spring (9 May), summer (19 July) and autumn (14 September), respectively to measure mineral + N (NH4N, NO 3 N) concentrations using a continuous-flow ion auto-analyser (Scalar SANplus segmented flow analyser, the Netherlands). Two-Way ANOVA and Duncan s Multiple-Range Test were used to compare the differences in soil NO 3 N and NH + 4N concentrations between treatments and soil depths for different seasons. To determine root N concentration, living roots taken from both control and N-treated plots using soil-sampling appliance (30 cm in length, 12 5 cm in diameter) on 9 May 2004 were washed and dried up in an oven. Root N concentrations were measured colorimetrically by the Kjeldahl acid-digestion method with an Alpkem auto-analyser (Kjektec System 1026 distilling unit, Sweden) after extraction with sulphuric acid. Results EFFECTS OF N FERTILIZATION ON SOIL WATER CONTENTS There were substantial fluctuations in soil water contents for both the control and the N treated plots. Temporal changes in soil water contents in the three soil layers of 0 10, and cm followed the similar trajectories, and soil water contents did not exhibit significant differences between control and treatment (P > 0 05) (Fig. 1). Further, there were no clear differences in soil water contents among the control plots and the N treated plots at the 0 10 cm soil layer in the two growing seasons, but soil water content in the N-fertilized plots was consistently lower than that in the control plots at the two deeper soil layers in 2004 throughout to the earlier growing months of No correlations between daily precipitation and soil water content were found (Fig. 1). Note the data for the daily precipitation obtained from the meteorological station of Duolun County were collected from the site 80 km away from our experimental site. Therefore, those data may not genuinely reflect the daily precipitation in our experimental site. EFFECTS OF N ADDITION ON SOIL CONCENTRATIONS Soil NO 3 N concentrations were not significantly different across the three soil layers in the control plots (Fig. 2). However, soil NO 3 N concentrations in summer (9 July 2004) were approximately two times higher than those in spring (9 May 2004) and autumn (14 September 2004) in the control plots (Fig. 2). There were marked increases in soil NO 3 N concentrations in the N added plots in May 2004, with the maximum occurring in the topmost soil layer (Fig. 2). In July and September 2004, soil NO 3 N concentrations in the top two soil layers in the N added plots became comparable to those in control plots, while those in the deeper soil layer (20 30 cm) in the N treated plots were significant greater than in the control plots (Fig. 2). In addition, NH + 4N concentrations were not significantly different between control and the treatment in the three soil layers (P > 0 05) (data not shown). N EFFECTS OF N ADDITION ON LIFE SPAN OF ROOTS IN DIFFERENT SOIL LAYERS NO 3 The survival rate of roots in the three soil layers in the control and N treated plots was shown in Fig. 3. In the control plots, the roots in the three soil layers exhibited comparable survivorship (Fig. 3). No significant differences in the mortal risk of

4 586 W.-M. Bai et al. Fig. 2. Soil N concentrations (mg kg 1 ) at the soil layers of 0 30 cm in spring (9 May), summer (19 July) and autumn (14 September) for control and N fertilization treatments in Different letters given in the top of error bars indicate significant difference at P < 0 05 levels (± SE, n = 5). NO 3 Table 1. Root longevity under the control and N fertilization treatments at soil depths of 0 10, and cm (mean ± SE) Soil depth (cm) Control N fertilization ± 5 a 59 ± 4 b ± 9 a 86 ± 9 a ± 14 a 59 ± 9 b ± 4 a 64 ± 3 b Mean longevity estimated by the Kaplan Meier survival analysis approach. Longevity differences between the control and N fertilization were compared using Log-rank test. In each horizontal row, the values with same superscript letters are not significantly different from each other at P = Fig. 1. Seasonal changes in soil water contents in control and N addition at the soil depths of 0 10, and cm in the growing seasons of 2004 and Data are mean ± SE for five replicates. Data of daily precipitation for 2004 and 2005 obtained from the local the meteorological station was given in the lower panel. roots present in the three soil layers as revealed by the Cox proportional hazards regression (Table 2). Roots in the topmost and deepest soil layers displayed a lower survivorship in response to N addition, while no effect of N addition on survivorship for roots in the soil layer of cm was observed (Fig. 3). The overall survivorship for roots in the three soil layers pooled as a whole was also reduced under conditions of N addition (Fig. 3). Accordingly, life span of roots in the three soil layers in the control plots was not significantly different (Table 1), but N addition significantly reduced life span from 78 to 59 days and 86 to 59 days for roots in soil layer of 0 10 and cm, respectively. The life span was not significantly different (P > 0 05) for roots present in the topmost soil layer in response to the N treatment. Further, the life span for roots in the three soil layers pooled as a whole was 81 days, and this value was reduced to 64 days in response to N addition (Table 1). To further characterize the overall mean root life span, we estimated values for duration with 25%, 50% and 75% of roots being dead, the corresponding values were 7, 13 and 46 days, respectively for the control plots. The Cox proportional hazards regression demonstrated that the N addition increased the mortal risk of roots present in the three soil layers as a whole by 122% (Table 2).

5 Root lifespan of Leymus chinensis and N addition 587 was reduced by 34% and 45%, respectively, in comparison with the roots born in spring, while the mortal risk of roots born in summer for N addition was increased 197% in comparison with those roots born in spring (Table 2). In the control plots, Log-rank test revealed that life spans for roots born in the three seasons differed significantly in the three soil layers, with life spans for spring-born and autumnborn roots being shortest and longest, respectively (Table 3). In addition to the root-born seasons, root life spans were also closely dependent upon the soil depths. For the spring-born roots, life span of roots in the topmost soil layer was significant shorter than that of roots in the two deep soil layers (Table 3). The summer-born roots in the deepest soil layer had significantly longer life span than those in the top soil layers (Table 3). By contrast, life span of roots born in autumn was relatively independent of soil depth (Table 3). Life span of the autumn-born roots was significantly longer than that of the spring-born roots in the each soil layer consistently (P < 0 001) in the N added plots (Table 3). N addition markedly reduced life span of the summer-born roots in all the three soil layers, while it shortened life span of the autumn-born roots in the topmost soil layer exclusively (Table 3). By contrast, for the spring-born roots, there was an increase and decrease in life span of the spring-born roots in the topmost and deepest soil layers respectively in response to N addition, but N addition had no effect on life span of the spring-born roots in the mid soil layer (Table 3). Effect of N addition on root production Fig. 3. Root survival curves under the control and N fertilization treatments at soil depths of 0 10, 10 20, and 0 30 cm during the growing seasons of Data shown in the figure were based on a total number of 1018 and 1623 individual roots for control and N fertilization respectively. Curves and mean life spans were generated using the Kaplan Meier method. Survival and life spans differences between the control and N fertilization were compared using Log-rank test. EFFECTS OF N ADDITION ON LIFE SPAN OF ROOTS BORN IN DIFFERENT SEASONS Roots of L. chinensis produced in the three seasons (spring, summer and autumn) exhibited different survival rates such that root survival rates for roots born in spring were lower than those born in autumn in the three soil layers (Fig. 4). The Cox proportional hazards regression demonstrated that the mortal risk of roots born in autumn for control and N addition N addition enhanced production of roots in the top two soil layers, but it had no effect on root production in the deepest soil layer in 2004 (Fig. 5). When data for roots in the three soil layers were pooled together, a significant increase in root length production in response to N addition was observed in 2004 (Fig. 5). By contrast, no effect of N addition on root length production was found in 2005, regardless of soil layers (Fig. 5). In addition, a highly positive correlation between root mortality and root length production in the three soil layers as a whole was found in both 2004 and 2005 (Fig. 5). RELATIONS OF ROOT LIFE SPAN WITH C ALLOCATION AND N ACQUISITION IN ROOTS The response of changes in root longevity to N addition was further characterized by plotting root longevity against root N concentration, soil NO 3 N concentration and maximum above-ground biomass, respectively. As shown in Fig. 6, across different plots in the two growing seasons, root longevity was negatively correlated with root N concentration, soil NO 3 N concentration and maximum above-ground biomass (P < 0 05). Discussion In the present study, we found that 91 7% of L. chinensis roots died within 1 year and the overall mean life span for roots

6 588 W.-M. Bai et al. Table 2. Results of the Cox proportional hazards regression on roots of L. chinensis, analysed in relation to the covariates including treatment, soil depth and time of root birth Covariate Parameter estimate SE Hazard ratio 95% CI Lower Upper P-value Treatment Ref. = Control (CK) N fertilization (+N) < Soil depth (CK) Ref. = 0 10 cm cm cm Soil depth (+N) Ref. = 0 10 cm cm cm Root birth (CK) Ref. = Spring Summer < Autumn < Root birth (+N) Ref. = Spring Summer < Autumn < Hazard ratios for categorical covariates are the risk of death relative to a reference level, given for each covariate in the left-hand column. A negative parameter indicates that increases in the covariate would result in decreases in the risk of mortality, and the opposite trend reflects positive parameter values. The values for 95% confidence intervals are upper and lower limits and are not symmetrical about the hazard ratio. P < 0 05 is considered to be significant. Fig. 4. Survival curves of roots born in spring, summer and autumn in different treatments and soil depths. Data were based on the total numbers of 1032, 847 and 762 individual roots born in spring, summer and autumn, respectively. The Kaplan Meier method was used to generate curves and to estimate mean life span. Survival differences between the timing of root birth were compared using Log-rank test.

7 Root lifespan of Leymus chinensis and N addition 589 Table 3. Longevity of roots born in spring, summer and autumn in different treatments and soil depths (mean ± SE). The Kaplan Meier method was used to estimate mean life span Treatment Soil depth (cm) Spring Summer Autumn Control ± 2 a 70 ± 10 b 147 ± 10 c ± 7 a 60 ± 14 b 151 ± 17 c ± 14 a 153 ± 41 b 101 ± 27 b N addition ± 6 a 24 ± 3 b 113 ± 8 c ± 6 a 35 ± 10 a 148 ± 16 b ± 9 a 34 ± 9 a 164 ± 30 b Longevity differences between the timing of root birth were compared using Log-rank test. In each horizontal row, the values with same superscript letters are not significantly different from each other at P = across the soil layers of 0 30 cm was 81 days. This value is comparable to root life span of and days for Festuca rubra grown in heterogonous and homogenous habitats, respectively (Partel & Wilson 2001). Several possible explanations may account for the relatively short root life span of L. chinensis. These include L. chinensis as a clonal plant with rhizome, the dominance of fine roots in L. chinensis, low annual temperature with relatively short growing season, and low soil moisture in the L. chinensis dominated steppe in Inner Mongolia. Furthermore, the relative short root life span in L. chinensis may also result from the shorter intervals (10 days) for monitoring root dynamics than others reported in the literature (cf. Gill et al. 2002; West et al. 2003). The short monitoring intervals would ensure that roots with rapid turnover are not lost, thus allowing the root life span to be more accurately estimated. The short life span found in the present study may result from artifact associated with methods used for distinguishing living and dead roots as living roots of some grass species grown in nutrient-poor environments may become dark brown. However, in the present study, the assumed dead roots based on the changes in root colours and morphology accounted for < 5% of the overall dead roots used for calculations of root life span, while > 95% of the overall dead roots were those that were lost during the imaging collection intervals. Therefore the short root life span for L. chinensis is unlikely to be accounted for by the method used for identifying dead roots. In addition, we can also discount the possibility that the observed short root life span is due to the studied soil surface being dried out as the soil water content in the top soil layer was not lower than that in the deep soil layers (Fig. 1). As a clonal grass, rhizome in L. chinensis may play an important role in conservation of nutrients and carbohydrates, thus roots in L. chinensis may mainly involve absorption of water and nutrients. The shorter root life span would facilitate root turnover and generation of young root, which in turn would favour water and nutrient use efficiency, thus allowing L. chinensis to better cope with their stressed habitats. Physiologically, rhizomes are strong sinks to compete for Fig. 5. The cumulative root length production in different soil depths in control and N added plots during growing seasons of 2004 and 2005 (Top two panels), and relationship between cumulative root mortality and root production (Lower panel). The root production and root mortality data were collected during 11 May and 23 September 2004 and 3 May and 12 September 2005, respectively. The differences in cumulative root production between control and N addition were examined by student s t-test, and the different letters given in the top of error bars indicate significant difference at P < 0 05 levels. The simulation models are y = 0 891x and y = 0 948x 0 199, respectively. photoassimilates with roots. As a result, it is conceivable that clonal plants may have lower carbohydrate contents in roots than nonclonal plants. This may also contribute to the observed short root life span in L. chinensis as low C input into roots reduced root life span (Farrar & Jones 2000). Longer root longevity in deeper soils has been found in grasses (Arnone et al. 2000) and trees (Kosola, Eissenstat & Graham 1995; Wells, Glenn & Eissenstat 2002; Anderson

8 590 W.-M. Bai et al. Fig. 6. Regressions of mean root longevity on root N concentration (a), soil NO 3 N concentration (b) and above-ground biomass (c). The data for mean root longevity were those mean values in the experimental plots across the three soil depths. The simulation models are y = 16 87x ; y = 7 116x and y = 0 176x , respectively. et al. 2003; Baddeley & Watson 2005). However, we found that root longevity of L. chinensis was relatively constant along the soil profiles up to 30 cm (Table 1). A similar independence of root mortality on soil depth has been reported in perennial bunchgrass Bouteloua gracilis in the shortgrass steppe (Gill et al. 2002). Our findings that the autumn-born roots of L. chinensis exhibited long life spans are consistent with those reported in other studies (Anderson et al. 2003). Differences in the temporal variability of root mortality have been ascribed to a number of factors, including soil water potential, temperature and the content of root carbohydrates (Bates, Dunst & Joy 2002; Anderson et al. 2003; Baddeley & Watson 2005). López et al. (2001) speculated that seasonal patterns in root life span are likely to be caused by varying root absorptivity in different seasons. The shortest life span for those roots born in spring could be primarily attributed to their physiological functions of roots born in different seasons. For instance, roots born in spring mainly function in absorbing water and nutrients, while roots born in autumn mainly perform functions of storing nutrients and producing new lateral roots. A primary factor determining root longevity is the quantity of carbohydrates transported into roots (Farrar & Jones 2000; Anderson et al. 2003). Lower carbohydrate contents in roots increase root mortality and reduce root longevity (Farrar & Jones 2000; Anderson et al. 2003). Previous study showed that individual plants of L. chinensis in spring allocate more C to the above-ground part during the vegetative stage, leading to lower C allocation to below-ground and decreased carbohydrates in roots (Zhang et al. 2006). These may account for the shorter root life span of L. chinensis in spring found in the present study. We found that N addition had significantly reduced the mean root longevity and survival rate of L. chinensis (Fig. 3 and Table 2). These findings are in good agreement with that reported by Van der Krift & Berendse (2002). Although there have been numerous studies focusing on root survivorship and root longevity (Eissenstat, Wells & Yanai 2000), few studies have directly investigated effects of N addition on root longevity. Rather, in many studies, researchers compared the root longevity for plants grown in N-rich and -poor habitats (West et al. 2003). To the best knowledge of authors, the present study is the first one to characterize root survivorship and root longevity in response to N addition in grass species. Longer root life span in nutrient-low habitats represents a strategy by which plants conserve nutrients by reducing root death (Van der Krift & Berendse 2002). On the other hand, higher root N concentration due to N addition may accelerate root respiration and deplete carbohydrates in roots, leading to a rapid root turnover and shortened root life span (Burton et al. 2000; Eissenstat et al. 2000). In response to enhanced soil nutrient resources, plants allocate relatively more C to the above-ground part; this would lead to shortened root life span due to a reduction of C accumulation in roots. Further, our data also revealed that N addition not only enhanced root mortality, it also stimulated root length production (cf. Fig. 5). Ruess, Hendrick & Bryant (1998) reported a similar positive correlation between root mortality and root length production. There were negative correlations between root longevity both with N concentration in roots and with soil NO 3 N concentration, and with the maximum above-ground biomass across all the plots (control and N added plots) in our 2-year study (Fig. 6), suggesting that increased N uptake and decreased C allocation in roots are likely to be the major mechanisms underlying the reduced root life span for L. chinensis in the N added plots. Furthermore, it should be noted that N addition caused the largest reduction in life span for roots

9 Root lifespan of Leymus chinensis and N addition 591 born in summer and in the deepest soil layer (Fig. 4). A further analysis reveals that N concentration was the highest in summer and in the deepest soil layer of all the other seasons and soil layers in the N fertilized plots except that in the surface layer in spring, which further corroborates the above mechanisms. It is evident that N leaching had occurred along the soil profile due to the sandy soil texture with high infiltration rate, with N added in September 2003 in the surface layer having been transferred to the deepest layer in the next summer (Fig. 2). In addition, soil water content was also consistently lower in the N added plots than in the control plots in the deepest soil layer in the two growing seasons of , which could also have contributed to the shortened life span in this case. In conclusion, we demonstrated that N addition led to a marked reduction in life span for roots of L. chinensis born in summer regardless of soil layers, while it increased life span of spring-born roots in the topmost and autumn-born roots in the deeper soil layer, respectively. These findings that longevity characteristics of L. chinensis roots responded to N addition spatially and temporally may be of ecological significance for understanding N use efficiency in native Inner Mongolia steppe. Acknowledgements We would like to thank S. H. Song, X. M. Zhan, H. Y. Wang and X. Li for their help in field and laboratory work. We thank Dr Shiqiang Wan for discussion and help in preparing the manuscript. This research was supported by the State Key Basic Research Development Program of China (No.2007CB106800), the Knowledge Innovation Major Project of CAS (No.KZCX2-XB2-01) and an innovative group research grant from the National Natural Science Foundation of China (No ). 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