Insights into the Nutritional Physiology of Nickel

Nickel (Ni) is essential for plants, yet its physiological role is poorly understood. Ni-deficient and Ni-sufficient pecan [Carya illinoinensis (Wangenh.) K. Koch] trees were compared regarding the impact of Ni nutritional status on reduced nitrogen (N) forms present in xylem sap at spring bud break. High performance liquid chromatography (HPLC) of xylem sap of Ni-sufficient trees found organic reduced N-forms to be primarily ureides (73%; citrulline > xanthine > ureidoglycolate > allantoic acid ≈ allantoin ≈ uric acid ≈ urea), followed by amide-N (26%; asparagine), and amino-N (1%; tryptamine and β-phenylethylamine). Nickel deficiency reduced xylem sap concentration of xanthine, asparagine, and β-phenylethylamine, yet greatly increased citrulline and allantoic acid. These data indicate that pecan is likely a ureide-N transporter and that Ni deficiency potentially disrupts ureide catabolism and urea cycle functionality; thus, potentially disrupting normal N-cycling during early spring when N reserves are being remobilized to sinks. INTRODUCTION Nickel (Ni) is an essential plant nutrient sometimes deficient in perennial orchard or nursery crops (Wood et al., 2004a, b, c, 2006; Ruter, 2005). Severe Ni deficiency in woody perennials can trigger growth disorders (i.e., “mouse-ear” or “little-leaf”) and an orchard replant disorder (Wood et al., 2004a, c; Ruter, 2005). Deficiency also disrupts nitrogen (N) and carbon (C) metabolism (Bai et al., 2006) in expanding foliage; although, the influence of Ni deficiency on N-forms translocated in spring xylem sap to canopy sinks is unknown. Such relationships are potentially important to management decisions pertaining to pollution, cost, and productivity of agricultural ecosystems. We hypothesize that endogenous Ni nutritional status quantitatively affects the reduced N-forms present in the xylem sap of pecan at spring bud break. MATERIALS AND METHODS The primary amide-N and ureide-N forms present in early spring xylem sap was determined by HPLC. Sap was collected and analyzed at equivalent bud break stages (inner bud-scale split of >50% of primary apical buds) from trees reflecting either of two distinct Ni nutritional states (i.e., deficient and sufficient). Three-year-old greenhouse seedlings were well managed regarding essential mineral nutrient elements, showing no visual symptoms of elemental deficiency except for Ni. Nickel sufficient trees (Ni-S) had prior season July leaf levels of 1-4 μg Ni·g dry weight, which is in the “sufficiency” range for Ni in pecan (Nyczepir et al., 2006). The Ni deficient (Ni-D) class exhibited classic morphological symptoms of Ni deficiency (Wood et al., 2004a) the previous growing season and again at bud break. July foliar Ni concentration of Ni-D trees was 0.004-0.540 μg Ni·g dry weight. The two Ni classes were achieved by growing trees on a Tifton Loamy Sand soil, which often causes morphological Ni deficiency symptoms in associated pecan orchards. Thus, one population of seedling trees exhibited classic morphological symptoms of severe Ni deficiency, and the second population exhibited no symptoms. Specimens from each of the two Ni status classes were randomly chosen for study. All trees received supplemental N as ammonium nitrate and N was not limiting. Proc. XXVII IHC Enhancing Econ. & Environ. Sustain. of Fruit Prod. in a Global Econ. Ed.-in-Chief: J.W. Palmer Acta Hort. 772, ISHS 2008 366 Xylem sap samples were taken from trees at bud break in late March. Sap from the Ni-D and Ni-S classes was collected by vacuum extraction from stems severed above the root collar and again just below the apical tip. The bark and phloem at the base of severed stems was removed to exclude phloem sap and the base placed under vacuum and the xylem sap dripped into 2 ml vials; thus, sap composition is a mix from nearly the entire length of the trunk of the seedlings. Equivalent volumes of xylem sap for each sample were thawed and twice centrifuged (20,000 g) for 30 min. The supernatant was further purified by removing molecules ≥ 10-kD by twice filtering through a Centricon-10 filter (Millipore Filter Units, Millipore, Bedford, MA) after centrifugation (5,000 g) for 75 min. The purified samples were then analyzed for ureides using UV spectroscopy and HPLC using two different types of columns and mobile phases. HPLC analysis and sample purification was according to Bai et al. (2006). RESULTS AND DISCUSSION HPLC analysis found early spring xylem sap comprised of at least seven ureido-N substances (allantoic acid, allantoin, citrulline, uric acid, urea, xanthine, and ureidoglycolate), one amide-N (asparagine) and two amino-N substances (tryptamine, and β-phenylethyamine) (Table 1). When viewed collectively within the context of Ni-S trees, the relative abundance of individual N forms was: citrulline > asparagine > xanthine > ureidoglycolate > allantoic acid > β-phenylethylamine > allantoin ≈ urea ≈ tryptamine ≈ uric acid. Allantoin, urea, uric acid, and tryptamine were present at trace levels. Intermediates of ureide catabolism were such that Ni deficiency reduced xanthine and allantoic acid concentration (Table 1); although concentrations of ureidoglycolate, allantoin, uric acid, and urea were unaffected. Ni deficiency also influenced urea cycle intermediates in that the concentration of asparagine declined and citrulline increased (Table 1, Fig. 1). Ni deficiency also reduced the xylem sap concentration of β-phenylethylamine (Table 1). Higher plants primarily transport organic N as either amide-, aminoor ureide-N forms (Schubert and Boland, 1990). Ureides are relatively high in dormant roots of many woody perennial species, where root export is via ascending xylem sap to active growing tissues and organs where the organic reduced-N is metabolized into proteins and nucleic acids (Schubert and Boland, 1990). These organic reduced-N atoms are predominately incorporated into ureides in pecan. The presence of several ureide pathway intermediates in xylem sap is circumstantial evidence that ureido-N catabolism likely occurs within the xylem stream while ascending to growing points during early spring. If so, then spring xylem sap composition differs depending upon proximity to sinks (e.g., shoots) or sources (e.g., roots). Ni deficiency quantitatively altered xylem sap reduced-N composition, but had no qualitative influence; thus, being similar to that observed in active growing Ni deficient pecan foliage (Bai et al., 2006). The decline of xanthine, and rise in allantoic acid, in sap of Ni deficient trees is evidence that Ni directly or indirectly affects ureide catabolism at one or more metabolic points, as was similarly found for ureide catabolizes in actively growing spring foliage (Bai et al., 2006). This raises the possibility that Ni ions can potentially influence the activity of certain ureide catabolic enzymes. Nickel deficiency also appears to disrupt the urea cycle. This is based on the observed increase in citrulline concentration and reducing asparagine concentration of asparagine in spring xylem sap (Table 1, Fig. 1). This metabolic shift indicates that Ni deficiency likely reduces either the presence or activity of argininosuccinate syntheses, which catalyzes citrulline to argininosuccinate (Fig. 1). It is concluded that pecan trees predominately transport N as ureides in early spring and that tree Ni nutritional status affects both ureide and amide metabolism, and xylem sap composition; and that Ni deficiency a) quantitatively affects sap composition of xanthine, allantoic acid, asparagine, citrulline, and β-phenylethylamine in spring xylem sap, b) alters the N:C composition of reduced-N components and therefore likely alters