Seasonal changes in tannin and nitrogen contents of Casuarina equisetifolia branchlets

Seasonal changes in tannin and nitrogen contents of Casuarina equisetifolia branchlets 1 first-author 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:linym@xmu.edu.cn †E-mail:linym@xmu.edu.cn linym@xmu.edu.cn 1 1 MOE Key Lab for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361005, China MOE Key Lab for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361005, China Fujian Academy of Forestry, Fuzhou 350012, China Fujian Academy of Forestry, Fuzhou 350012, China Seasonal dynamics of total phenolics (TP), extractable condensed tannins (ECT), protein-bound condensed tannins (PBCT), fiber-bound condensed tannins (FBCT), total condensed tannins (TCT), and protein precipitation capacity (PPC) in young, mature and senescent branchlets of Casuarina equisetifolia were studied at Chishan Forestry Center of Dongshan County, Fujian Province, China. In addition, nitrogen contents of branchlets at the different developmental stages were also determined. The contents of TP and ECT, and PPC in young branchlets were significantly higher than those in mature and senescent branchlets through the season. However, PBCT contents were significantly higher in senescent branchlets than those in young and mature branchlets; FBCT fluctuated with season. Young branchlets had the highest N content, which decreased during branch maturity and senescence. The highest contents of TP and the lowest contents of TCT and N in young and mature branchlets were observed in summer. There was a significant negative correlation between TP and N contents. In contrast, TCT contents were positively correlated to N contents. Nutrient resorption during senescence and high TCT:N ratios in senescent branchlets are the important nutrient conservation strategies for C . equisetifolia . Casuarina equisetifolia Condensed tannins Total phenolics Nitrogen Seasonal dynamics INTRODUCTION 1 Casuarina equisetifolia is a nitrogen-fixing tree of considerably social, economic and environmental importance in tropical/subtropical littoral zones of Asia, the Pacific, and Africa. It is commonly used in agro-forestry systems for soil stabilization and reclamation work and in coastal protection and rehabilitation (Pinyopusarerk and House, 1993 1993 ; Pinyopusarerk and Williams, 2000 2000 ). It is one of the most extensively introduced tree species outside its natural range. There are more than 300 000 ha of C . equisetifolia plantations in the coastline of southern China (Zhong et al., 2005 2005 ). Despite the widespread planting and known ecological and physiological properties of C . equisetifolia , very little has been done to explore secondary metabolism production along with seasonal changes, although they are recognized as high in tannin and astringent (Okuda et al., 1980 1980 ). Tannins play a role in a number of ecological processes in addition to herbivore defense, including litter decomposition, nutrient cycling, nitrogen sequestration, microbial activity, humic acid formation, metal complexation, and pedogenesis (Kraus et al., 2003 2003 ). Tannins comprise a significant portion of terrestrial biomass C (Hernes et al., 2001 2001 ). Leaves and bark may contain up to 40% tannin by dry weight (Kuiters, 1990 1990 ; Matthews et al., 1997 1997 ; Lin et al., 2006 2006 ; 2007 2007 ), and in leaves and needles tannin contents can exceed lignin levels (Hernes et al., 2001 2001 ). Because tannins are complex and energetically costly molecules to synthesize, their widespread occurrence and abundance suggest that tannins play an important role in plant function and evolution (Cates and Rhoades, 1977 1977 ; Zucker, 1983 1983 ). Some species rich in polyphenols appear to gain a competitive advantage by altering nutrient cycling and thus influencing forest dynamics (Northup et al., 1998 1998 ; Fierer et al., 2001 2001 ; Kraus et al., 2003 2003 ). Plants greatly influence soil processes through litter inputs (Aber et al., 1990 1990 ); thus, alterations in foliar chemistry have important implications for plant-litter-soil interactions and ecosystem function (Kraus et al., 2004 2004 ). Traditionally, justification for the high metabolic cost associated with the production of tannins was attributed to improve herbivore defense (Feeny, 1970 1970 ). Two major hypotheses have been proposed to predict the effects of environmental factors on secondary metabolite concentrations. The carbon-nutrient balance (CNB) hypothesis postulates that phenolic levels in plants are determined by the balance between carbon and nutrient availability (Bryant et al., 1983 1983 ). The growth-differentiation balance (GDB) hypothesis (Loomis, 1932 1932 ; Lorio, 1986 1986 ; Herms and Mattson, 1992 1992 ) considers factors that limit growth and differentiation (the sum of chemical and morphological changes that occur in maturing cells, including carbon-based secondary synthesis). The production of phenolics dominates when factors other than photosynthate supply are suboptimal for growth (e.g., under nutrient limitation or moderate drought). Resource-based theories assume that the synthesis of defensive compounds is constrained by the external availability of resources and internal trade-offs in resource allocation between growth and defense (Riipi et al., 2002 2002 ). They state that growth processes dominate over the production of defensive compounds, and that more carbon is left for defensive compounds only when plant growth is restricted by a lack of mineral nutrient (emphasized by the CNB hypothesis) or by any factor (according to the GDB hypothesis) (Haukioja et al., 1998 1998 ). Jones and Hartley ( 1999 1999 ) presented a protein competition model (PCM) for predicting total phenolics allocation and content in leaves of terrestrial higher plants. They stated that “protein and phenolics synthesis compete for the common, limiting resource phenylalanine,” so nitrogen (N) rather than C is the limiting resource for synthesis of phenolics. Despite the growing knowledge of the physiological basis and ecological consequences of leaf phenolics presence in plant tissues, only a few extensive datasets are available to evaluate the relative importance of leaf phenolics variability caused by environmental changes and developmental stages (Covelo and Gallardo, 2001 2001 ; Lin et al., 2007 2007 ). This variability may determine not only the susceptibility of plants to herbivore attack, but also important aspects of nutrient cycling in terrestrial and aquatic ecosystems (Gallardo and Merino, 1992 1992 ; Serrano, 1992 1992 ; Mafongoya et al., 1997 1997 ; Northup et al., 1998 1998 ). According to PCM, the growth-defense trade-off depends not only on competition for a limited pool of available carbohydrates, but also on competition for nitrogen as a component of common precursor compounds (Jones and Hartley, 1999 1999 ; Gayler et al., 2007 2007 ). In this regard, information on the seasonal dynamics of tannins and nitrogen contents is necessary. The objective of the present study was to test the validity of the following hypotheses: (1) Tannins of branchlets at different developmental stages follow a seasonal pattern; (2) The production of phenolics dominates under nutrient limitation. To answer these hypotheses, a field investigation of C . equisetifolia was conducted at Chishan Forestry Center of Dongshan County, Fujian Province, China. MATERIALS AND METHODS 1 Study areas 2 The study was carried out at Chishan Forestry Center of Dongshan County (23°40′ N, 117°18′ E), Fujian Province, China. The climate of the region belongs to southern subtropical maritime monsoon climate, with annual temperature ranging from 3.8 °C to 36.6 °C. Mean annual precipitation and evaporation are 1 103.8 mm and 2 027.9 mm, respectively. The rain season is from May to September and the dry season is from October to April of the next year. The soils are coastal sandy barren. The C . equisetifolia plantations were artificial, pure forests that were planted in 1999. The coverage of dense forest was 0.7, tree density was 3 552 tree/ha, canopy height ranged from 5~7 m, and diameter at breast height (DBH) was (8.1±1.4) cm. The average N and P contents of the soils at 20 cm depth were (54.49±7.89) and (2.69±0.42) mg/kg, respectively, and soil pH was 4.19±0.05. Materials 2 Thirty individuals of C . equisetifolia were chosen and labeled. The height and growth conditions of the chosen trees were similar. The development stages of branchlets were demarcated into three stages, i.e., young branchlets (newly-emerged, usually shorter than 5 cm in length at the top of branch, and light green in color), mature branchlets (fully developed, usually 15~25 cm long, and dark green in color), and senescent branchlets (old branchlets, white or grey in color). Branchlets damaged by insects and disease or mechanical factors were avoided. Young, mature and senescent branchlets of each labeled tree were collected in March, June, September, and December of 2007. All samples were taken to the laboratory immediately after sampling and cleaned with distilled water. Chemical analyses 2 All chemicals were of analytical reagent purity grade. An additional standard denoted here as purified tannin, was extracted from C . equisetifolia branchlets and purified on Sephadex LH-20 (Amersham, USA) according to the procedure previously described by Asquith and Butler ( 1986 1986 ) as modified by Hagerman ( 2002 2002 ). The condensed tannin standard was freeze-dried and stored at −20 °C until required. Procedures described by Lin et al.( 2006 2006 ) were used to determine total phenolics (TP), extractable condensed tannins (ECT), protein-bound condensed tannins (PBCT), fibre-bound condensed tannins (FBCT), and protein precipitation capacity (PPC). TP were measured with the Prussian blue method (Graham, 1992 1992 ), and ECT, PBCT and FBCT were assayed by the butanol-HCl method (Terrill et al., 1992 1992 ) using purified tannins from C . equisetifolia branchlets as the standard. The contents of total condensed tannins (TCT) were calculated by adding the respective quantities of ECT, PBCT, and FBCT (Terrill et al., 1992 1992 ). A radial diffusion assay was used to determine the PPC (Hagerman, 1987 1987 ). N content was determined with Nessler’s reagent after Kjeldahl digestion of powdered samples with sulfuric acid and hydrogen peroxide (Mae et al., 1983 1983 ). Calculations 2 Resorption efficiency (RE) was calculated as the percentage of N recovered from the senescing leaves (Aerts, 1996 1996 ; Killingbeck, 1996 1996 ): RE (%)=( A 1 − A 2 )/ A 1 ×100, where A 1 is N content in mature branchlets; A 2 is N content in senescent branchlets. Statistical analyses 2 All measurements were replicated three times and analyzed using one-way analysis of variance (ANOVA) (SPSS 11.0 for Windows) with TP, ECT, PBCT, FBCT, TCT, and PPC. RESULTS 1 Seasonal dynamics of total phenolics contents 2 TP content was highest in young branchlets, (146.17±4.14) to (169.23±4.78) mg/g, and decreased with maturity and senescence. TP did not follow the identical pattern for young, mature, and senescent branchlets. TP contents of young [(169.23±4.78) mg/g] and mature [(118.40±5.81) mg/g] branchlets in summer were relatively high than those in other seasons. TP content of senescent branchlets was the lowest in summer [(24.56±0.92) mg/g] and the highest in winter [(98.74±16.10) mg/g] (Fig. 1 1 ). Seasonal dynamics of condensed tannin contents 2 ECT contents of young and senescent branchlets were significantly higher in winter than in other three seasons (Fig. 2a 2a ), which was different from the observation of ECT contents of mature branchlets. The ECT contents of young and senescent branchlets in winter reached (316.17±64.10) and (168.06±44.19) mg/g, respectively. ECT contents of mature branchlets ranged from (101.44±16.17) to (137.36±13.48) mg/g, and remained unchanged with season. ECT contents of senescent branchlets were significantly lower than those of young and mature branchlets except for the mature branchlets in winter, showing that the ECT contents of the branchlets decreased with senescence. PBCT contents were significantly higher in senescent branchlets than in young and mature branchlets (Fig. 2b 2b ), showing that PBCT contents increased with senescence. The highest PBCT contents of young and mature branchlets in autumn were (13.55±2.32) and (7.43±1.01) mg/g, respectively. PBCT contents of young and mature branchlets both increased gradually during the growing season and then declined in winter. However, FBCT contents of the branchlets showed the decreasing trends during the growing seasons, and then increased in winter (Fig. 2c 2c ). Except for in winter, FBCT contents of senescent branchlets were significantly higher than those of young and mature branchlets (Fig. 2c 2c ). PBCT was significantly higher than FBCT for senescent branchlets, but there was no significant difference for young and mature branchlets. ECT accounted for 89%~95% of TCT in young and mature branchlets, with 5%~11% being bound to protein or fibre (Fig. 2d 2d ). Seasonal dynamics of protein precipitation capacity 2 PPC was the highest in young branchlets, and decreased with maturity and senescence (Fig. 3 3 ). The PPCs of young [(530.19±30.39) cm 2 /g] and mature [(398.28±23.01) cm 2 /g] branchlets were lower in spring than in other seasons. Seasonal dynamics of nitrogen content and resorption efficiency 2 Young and mature branchlets had significantly higher nitrogen contents than senescent branchlets. The N contents of senescent branchlets were all below 0.86% (Fig. 4 4 ). The lowest N contents all occurred in the summer for young, mature, and senescent branchlets. The N contents remained relatively stable for the other seasons. Nitrogen resorption efficiency (NRE) ranged from (51.01±3.94)% to (63.00±8.61)% with no significant difference among four seasons. Numbers within the ellipses represent the nitrogen resorption efficiency DISCUSSION 1 Young branchlets had the higher contents of TP and ECT than mature and senescent branchlets. The observed changes in TP and ECT contents of the branchlets associated with different development stages are in accordance with the findings reported for different mangrove species (Lin et al., 2006 2006 ; 2007 2007 ) and oak species (Makkar et al., 1988 1988 ; Rossiter et al., 1988 1988 ). Young leaves need to be well defended, since damage to young leaves may cause a larger decline in future photosynthesis than damage to mature leaves (McKey, 1974 1974 ; Harper, 1989 1989 ). In addition, the fact that young branchlets contain much higher contents of TP and ECT compared with mature and senescent branchlets may indicate that young branchlets are experiencing more intense selective pressure than mature and senescent branchlets. Many of the soluble carbon compounds (which also include polyphenols) are expected to be translocated from leaves during senescence (Mafongoya et al., 1998 1998 ). The depletion of TP and ECT during senescence in the spring, summer and autumn and the enrichment of TP and ECT during senescence in winter were found in the present study. The decrease in phenolics content may reflect an active turnover of phenolics and an increase in bound or nonextractable phenolics during senescence (Fig. 2b 2b ). As polyphenols are water-soluble and susceptible to leaching (Hättenschwiler and Vitousek, 2000 2000 ), leaching of polyphenols or tannins by sporadic rain (from green leaves) might be a cause for the net ‘enrichment’ in senesced leaves (Teklay, 2004 2004 ), but the exact cause or mechanism is difficult to ascertain. Constantinides and Fownes ( 1994 1994 ) also found both an increase and a decrease in polyphenol contents. Kuhajek et al.( 2006 2006 ) observed that no significant effects of leaf age on both condensed tannins and total phenolics. As for bound condensed tannins, PBCT contents were significantly higher in senescent branchlets than in young and mature branchlets. Seasonal changes in leaf chemistry reflect changing demands for carbohydrates and nutrients resulting from normal growth and differentiation processes (Wareing, 1959 1959 ; Moorby and Wareing, 1963 1963 ). The GDB hypothesis (Loomis, 1932 1932 ; Lorio, 1986 1986 ; Herms and Mattson, 1992 1992 ) and the CNB hypothesis (Bryant et al., 1983 1983 ) assume that the synthesis of carbon-rich secondary chemicals is limited by the availability of photosynthesis (carbon). According to these hypotheses, growth processes dominate over differentiation or production of carbon-rich secondary compounds as long as conditions are favorable for growth, but the GDB hypothesis is associated with the temporal variation in growth activity more directly than the CNB hypothesis (Tuomi, 1992 1992 ). If plant growth is active, and therefore demands large amounts of carbon, allocation to carbon-rich secondary metabolites, e.g., phenolics, is predicted to decline; however, when growth is limited more than photosynthesis, allocation to defense will increase (Riipi et al., 2002 2002 ). Under the assumptions of the GDB hypothesis, allocation to phenolics should be low in spring during rapid growth of the short-shoot leaves (Riipi et al., 2002 2002 ). The amounts of phenolics should increase most quickly in summer, when the photosynthetic capacity of the newly matured leaves is highest (Mooney and Gulmon, 1982 1982 ). The highest TP content in summer was found in this paper (Fig. 1 1 ). C . equisetifolia growth is active from May to September (rain season) with the relatively high soil water availability. According to the PCM hypothesis, protein demand should be highest when plant grows rapidly, and allocation to those phenolics that are derived from phenylalanine should simultaneously decrease (Jones and Hartley, 1999 1999 ), as phenylalanine is the common precursor of either protein or condensed tannins synthesis (Hättenschwiler and Vitousek, 2000 2000 ). However, the second major group of phenolics, the hydrolysable tannins, has gallic acid as its precursor (Haukioja et al., 1998 1998 ). Therefore, depending on the relative strength of the synthetic route via dehydroshikimic acid to gallic acid, hydrolysable tannins may or may not trade off directly with protein synthesis (Haukioja et al., 1998 1998 ). Hydrolysable tannins are thought to be metabolically cheaper than phenylpropanoids, and the synthesis of it in the season may be a cost-saving defense strategy during a time when condensed tannins are not yet effective as defense (Haukioja et al., 1998 1998 ; Salminen et al., 2001 2001 ). However, the changes in contents do not necessarily reflect the quantitative allocation of tannins to the leaves, because of rapid turnover of labile compounds (Kleiner et al., 1999 1999 ) and because the contents are affected by concomitant changes in proportions of other components of the leaves, e.g., structural leaf components (Koricheva, 1999 1999 ). The lowest nitrogen contents of branchlets at the different development stages occurred in summer, when C . equisetifolia growth is active. Similarly, Aerts et al.( 1999 1999 ) suggested that summer warming reduced N contents of mature and senescent leaves in Rubus . First, a portion of N was allocated to other portions (e.g., roots and flowers); for example, the peak of the flowering period appears from April to June for C . equisetifolia (Morton, 1980 1980 ). Second, N contents were diluted by branchlets mass accumulation during summer when C . equisetifolia grew rapidly. Changes in leaf N contents may directly impact the photosynthetic capacity of the species involved, as there is usually a direct relation between leaf N content and the maximum rate of photosynthesis (Lambers et al., 1998 1998 ). In this study, TP contents were inversely related to N contents. It is common to find a negative correlation between N and secondary compound contents, such as phenolics and tannins (Horner et al., 1987 1987 ; Mansfield et al., 1999 1999 ). This pattern lends to support source-sink hypotheses, such as the CNB hypothesis (Bryant et al., 1983 1983 ) and the GDB hypothesis (Herms and Mattson, 1992 1992 ) that predict increased C allocation to secondary C compounds under low nutrient conditions. However, there was a positive correlation between N and TCT contents, which is consistent with a previous study (Horner et al., 1987 1987 ) but inconsistent with other study (Kraus et al., 2004 2004 ). They may result from differences in the carbon:nutrient balance of the plants resulting from differences in relative resource availability (Horner et al., 1987 1987 ). The young leaves were likely to produce toxic effects even at low levels of intake, and PPC was considered to be related to the biological activity (Martin and Martin, 1982 1982 ; Deshpande et al., 1986 1986 ) and was very high in the young leaves. The research on C . equisetifolia comes to the same result (Fig. 3 3 ). The capacity of tannin to bind proteins was related to the molecular size of the tannins (Makkar et al., 1987 1987 ). In general, it was found that the larger-sized condensed tannin could precipitate more protein than the smaller-sized condensed tannin (Osborne and McNeill, 2001 2001 ). In this study, there was no significant correlation between TP or TCT and PPC in young and mature branchlets (Table 1 1 ), which is consistent with the previous study (Martin and Martin, 1982 1982 ). However, there was a significantly positive correlation between TP or TCT and PPC in senescent branchlets. The results indicate that the possible increase in degree of polymerization with senescence leads to the more larger-sized tannins and the more protein precipitated. Branchlets TP vs N TCT vs N TP vs TCT TP vs PPC TCT vs PPC Young −0.615 ( P =0.033) 0.554 ( P =0.061) −0.081 ( P =0.802) 0.190 ( P =0.554) 0.242 ( P =0.448) Mature −0.785 ( P =0.002) 0.497 ( P =0.100) −0.126 ( P =0.695) 0.147 ( P =0.649) −0.647 ( P =0.023) Senescent 0.374 ( P =0.231) 0.264 ( P =0.407) 0.926 ( P =0) 0.870 ( P =0) 0.870 ( P =0) TP:N ratio was the highest in young branchlets and decreased with maturity and senescence in spring and summer, while TP:N ratio of branchlets decreased with maturity and increased with senescence in autumn and winter (Fig. 5a 5a ). TP:N ratios of young and mature branchlets were both the highest in summer, and remained relatively stable in other three seasons. The relatively low TP contents of senescent branchlets paired with high N contents in spring resulted in the low TP:N ratios. The TP:N ratios of senescent branchlets increased through the season (from spring to winter). Seasonal changes in TCT:N ratios during development stages were different from the observation of TP:N ratios (Fig. 5b 5b ). TCT:N ratios of senescent branchlets were significantly higher than those of young and mature branchlets. TCT:N ratios of senescent branchlets were significantly higher than TP:N ratios of senescent branchlets in corresponding seasons. The role that tannins play in soil processes is believed to occur largely through their ability to precipitate proteins, as well as their relative resistance to decomposition (Kuiters, 1990 1990 ; Kraus et al., 2003 2003 ). The amount of tannins entering the soil relative to the amount of proteins or N may be the key factor influencing soil nutrient cycling. Therefore, parameters such as TP:N and CT:N ratios may be the best predictors of litter quality (Kraus et al., 2004 2004 ). In green foliage, high TP:N and TCT:N ratios may help reduce herbivory. In our study, TP:N ratios in young branchlets were higher than those in mature branchlets. The highest levels of TP:N in young and mature branchlets both occurred in summer, indicating potentially increased grazer deterrence. TP:N ratios in senescent branchlets increased through the season (from spring to winter). TCT:N ratios were significantly higher in senescent branchlets than those in young and mature branchlets. Nutrient resorption (about 50%~60%) during senescence and high TCT:N ratios in senescent branchlets are the important nutrient conservation strategies for C . equisetifolia . References JD Aber , JM Melillo , CA McClaugherty . 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