RNase A in pecan xylem sap

Ribonuclease activity, molecular mass and amino acid sequence analysis identified a 14 kDa RNase A present as a component of the late winter xylem sap of a long-lived perennial tree crop at a time when the pecan tree is transitioning from dormancy to initiation of early spring growth. This is a time when the tree requires access to considerable amounts of chemically reduced N for growth of new tissues and organs in preparation for the upcoming growing season. The Ni nutritional status influences the concentration of xylem sap RNase A. A biochemical consequence of Ni deficiency in plants is a potential reduction in RNase A within spring xylem sap. This deficiency potentially limits N cycling and metabolism at a critical time, in which perennial plants are preparing for rapid growth, by impairing RNA catabolism in apoplastic space.

Activity of the pecan Ni-RNase A complex

It is of physiological significance that when pecan RNase A is exposed to Ni it exhibits ureolytic activity. The ureolytic activity of this xylem sap 14 kDa Ni-RNase A complex is similar to that of the bovine pancreatic Ni-RNase A complex, but only about 40% as great as that of jack bean urease. While there is a theoretically possibility that the purified xylem sap RNase A protein contained a urease contaminate, the rigor of the purification protocol is unlikely to have left such a contaminate. However, further study is required in order to definitively eliminate such a possibility. The results are suggestive that xylem sap RNase A plays a duel role in regards to early season N metabolism of perennial plants. In addition to a primary function of cleaving RNA, there is a putative secondary function of catabolizing urea in ascending xylem sap and associated apoplastic space. These activities are likely to vary as a function of sap Ni concentration. Whether or not this ureolytic activity actually occurs in planta remains to be determined. The presence of urea in the apoplast, from either urea uptake from soil by roots, or from catabolism of purine nucleotides, ureide pathway intermediates, or from arginine argues that small apoplastic proteins capable of converting urea to NH₄ might prove metabolically important, especially during the time of transitioning from dormancy to active growth.

The influence of Ni on RNase A

Our finding that the Ni-RNase A complex from sources as diverse as pecan and cattle exhibits substantial ureolytic activity naturally raises questions about its relevance to N metabolism naturally occurring in plants and animals. It appears that the nature of this ureolytic activity depends on cellular Ni status, which in turn should vary according to homeostatic processes and organismal exposure to Ni. Transition metal ions, such as Ni, elicit various enzymatic characteristics, including nucleophilic catalysis, electron transfer, and stabilization of protein structure. X-ray diffraction and three-dimensional structures of RNase A indicates the enzyme possess potential to bind Ni ions at His 105, where it acts as a nucleation site and causes a conformational change [19] capable of enabling significant ureolytic activity. Similarly, a plant urease from Kiebsilla aerogenes appears to possess a functional requirement for two Ni ions per active site [7], with Ni ions bound at Arg 70 and Gly 197 (amino acid residues) where Ni easily makes contact with urea substrate molecules [13].

Observations herein indicate that Ni binding to RNase A, normally a non-metalloenzyme, enables ureolytic activity; thus, the Ni-RNase A complex is functionally an alternative molecular form of urease, an essential Ni-metalloenzyme. This complex might therefore play a role in urea catabolism by perennial plant species, especially those like pecan that possess relatively high Ni requirements. A similar role might also exist in other plants and animals. While Ni possesses an essential metabolic role in plants and animals, that role is somewhat enigmatic. It now appears possible that in certain cases Ni could be influencing N cycling through its influence on RNase A. While many proteins can bind or contain Ni [20], this aspect of Ni nutritional physiology in has received little attention in planta or in animal.

Several enzyme systems in bacteria and lower plants require Ni. Examples include NiFe-hydrogenase, carbon monoxide dehydrogenase, acetyl-CoA decarbonylase synthase, methyl-coenzyme M reductase, superoxide dismutase, Ni-dependent glyoxylase, aci-reductone dioxygenase, and methyleneurease [1, 21, 22]. It is the activation of urease, to date, that is the most commonly recognized function of Ni in higher plants [22]. Ni can also replace Zn or Fe, and other transition metal ions, in certain other metalloenzymes of lower plants [1], and can probably do so to some degree in higher plants and animals. Circumstantial evidence indicates that ureide-transporting plant species, such as pecan, possess a higher Ni requirement than amide-transporting species [23]; thus, raising the possibility that ureide transporting species might possess enzymes, other than the standard ubiquitous urease, that require Ni for activation or for enhanced activity. Likely candidates are one or more enzymes affecting N metabolism; thus, raising the possibility, that RNase A possesses a dual role in cellular metabolism and that this role depends on endogenous bioavailability of Ni.

This potential ureolytic form of RNase A in plants and animals possibly affects N-metabolism via hydrolysis of either urea or RNA depending on Ni exposure. For example, in germinating rice seeds, Ni²⁺ exposure increases the levels of RNA by a decline in hydrolysis [24–26]; however, in eggplant ribonuclease activity is increased by Ni²⁺[26].

It appears that not only does bovine pancreatic RNase A potentially possess duel functionality as urease when exposed to Ni ions, but that a pecan orthologue also functions as an urease within the early spring xylem sap of pecan when N reserves are being mobilize from storage tissues to emerging sinks. Urea is a catabolic product of ureide catabolism, e.g., allantoate and ureidoglycolate [27–29]; thus, the xylem sap, and affiliated apoplast, of many long-lived perennials may be a location contributing to the conversion of urea to NH₄ in preparation for protein and nucleic acid anabolism because of imminent deployment of canopy organs. Both free and bound Ni readily transports in xylem vessels [30–32] and therefore potentially influences the nature of RNase A activity.

This discovery potentially broadens knowledge of possible secondary roles of RNase A in organisms in regards to N metabolism. Because several structural orthologues or paralogues of ribonucleases possess biological action [10, 11], it is possible that Ni binding to these proteins also alters their activity; thus, highlighting new possibilities for metabolically important roles for Ni in higher organisms.

Plant materials

Three-year-old pecan seedlings, originating from seed of open pollinated 'Desirable’ cv. trees, were grown in pots within a greenhouse to produce two Ni nutritional classes of trees—'Ni sufficient’ (Ni⁺) vs. 'Ni deficient’ (Ni^-), based on expression of morphological symptoms of Ni deficiency [11]. The Ni^- trees were produced from growing in a Tifton Loamy Sand soil. This soil often causes Ni deficiency symptoms in associated commercial pecan orchards. Ni⁺ trees from the same soil had prior season July leaf Ni concentrations of 1–4 μg^.g^-1 dry weight, which is considered to be within the 'sufficiency’ range for Ni in pecan. The Ni^- trees had Ni concentrations ≤ 0.8 μg^.g^-1 dry weight [33], which is low enough to trigger the appearance of easily visible morphology based symptoms.

Six specimens from each of the two 'Ni status classes’ were randomly chosen for study from a population of trees, based on expression of visual symptoms of Ni deficiency occurring at the time of spring bud break. Spring xylem sap was collected and analyzed from several trees exhibiting the two classes of Ni nutritional status. Collection was at bud break, with bud break defined as inner bud scale split of >50% of primary apical buds. Xylem sap was collected by vacuum extracted from stems severed at the root collar and again just below the apical tip; with the phloem and bark associated with the stem base peeled back to ensure that xylem sap was not contaminated with phloem sap. The base of severed stems was placed under vacuum and the exuding xylem sap dripped into 2 ml collection vials. Total Ni concentration of xylem sap of Ni^- and Ni⁺ trees was determined with xylem sap collected as described above, and quantified by ICP-MS [34].

Extraction and purification of xylem sap urease

The xylem sap samples were diluted in Buffer E [25 mM 2-morpholineethanesulfonic acid (Mes), pH 6.2, 2.5 mM MnSO₄, and 2.5 mM DTT] and centrifuged at 20,000 g for 1 h and 30,000 g for 1 h. This supernate was passed through a 0.22 μM membrane to remove impurities, and then twice extracted with chilled acetone (90%) to precipitate proteins. This precipitate was dissolved in Buffer E, with protein purification via ammonium sulfate fractionation, gel filtration chromatography and ion exchange chromatography according to previously reported methods [35]. Proteins were further purified by passing through ammonium sulfate fractionation (50%), filtering to exclude ≤ 10 kDa molecules (Amicon Centricon YM-10, Millipore Corporation, Bedford, Ma, USA), and finally via gel filtration (Superdex 200 10/300 GL column; controlled by ÄKTAbasic –Systems, Amersham Biosciences, Piscataway, NJ, USA) and ion exchange (Mono Q H/R 5/5 column; controlled by ÄKTAbasic –Systems) chromatography. Buffer E was used as solvents and NaCl was added into Buffer E for the salt gradient elution during purification [36]. Fractions with urease activity were pooled, desalted using a small P-10 column, and concentrated by centrifugation for 2 h at 5,000 g at 4°C with Amicon Centricon YM-10. The smaller molecules were removed because they contain phenolic substances that interfere with enzyme activity. Fresh purified enzyme was subsequently loaded on large (16 cm × 16 cm) gradient (8-16%) sodium dodecyl sulphate -polyacrylamide electrophoresis (SDS-PAGE) gels for purity analysis.

RNase A source and activity

RNase A from bovine pancreas (90 units/mg protein) came from the USIB Corporation (Cleveland, OH, USA). Its purity was examined with SDS-PAGE (10-20% gradient gel, 16 cm × 16 cm in size) while controlling temperature (at 12–15°C) during electrophoresis. Purified protein was denatured in SDS-gel sample buffer and electrophoresed on a SDS-10 to 20% polyacrylamide gradient gel. The nuclease activity of RNase A from bovine pancreas was verified by suspending RNA (ribonucleic acid from baker’s yeast, Saccharomyces cereviae; Sigma, St. Louis, Mo, USA) in Buffer E [25 mM 2-morpholineethanesulfonic acid (Mes), pH 6.2, 2.5 mM MnSO₄, and 2.5 mM DTT) [35]. RNase A (10 μL) was then added to the RNA suspension and the reaction mixture (2 mg/ml) incubated at 25°C for 1 h and then centrifuged (5,500_g at 4°C). The mixture was then filtered to exclude ≤ 50 kDa molecules (Amicon Centricon YM-50, Millipore Corporation, Bedford, Ma, USA). The fraction was rinsed with Buffer E, centrifuged (at 5,500_g at 4°C), and again filtered to exclude ≤ 50 kDa molecules. The > 50 kDa fractionation was suspended in Buffer E (2 ml) and the reaction monitored based on absorbance at 260 nm. Buffer E served as a blank and untreated RNA (2 ml) served as a positive control.

Assay of urease activity in protein solutions with molecular mass ≥10 kD

Urease activity was described by Kaltwasser and Schlegel [36], but with slight modification [37]. The assay is completed as a coupled enzyme with Glu dehydrogenase. All chemical assay reagents were dissolved in 0.1 M potassium phosphate buffer (pH 7.6). The assay mix was 0.37 ml of 0.1 M potassium phosphate buffer (pH 7.6), 0.1 ml of 1.8 M urea, 0.1 ml of 0.025 M ADP, 0.2 ml of 0.008 M NADH, 0.1 ml of 0.025 M α-ketoglutarate before adding 0.1 ml of 50 units/ml Glu dehydrogenase and 5 μL of enzyme solution. The change in A₃₄₀ at 25°C was recorded at 0.5, 1, 3, and 5 min. Urease of jack bean (Canavalia ensiformis; 29.5 units/mg; Sigma, St. Louis, Mo, USA) was used as a reference. A unit is defined as the amount of urease causing oxidation of 1 μM of NADH/min at 25°C, pH 7.6, in a coupled reaction using Glu dehydrogenase. Protein concentration was determined with the Bio-Rad protein assay with bovine serum albumin as standard.

Effect of nickel ions on urease activity of RNase A

The purified RNase A solution (10 μl), either from bovine or pecan sap, was mixed with 10 μl of Ni-nitrate solution at different concentrations (0, 333, 1,000, and 3,300 μM, respectively) in a cuvette to give a final Ni concentration of 0, 0.0033, 0.0100, and 0.0333 μM/ml. After 2 min, 998 μl of chemical substrate solution was added and mixed, with measurement of urease activity determined as described above.

Determination of the N-terminal amino acid sequence

Purified urease from pecan sap was denatured in SDS-gel sample buffer and electrophoresed on an SDS-10 to 20% polyacrylamide gradient gel and then the protein was transferred to a PVDF membrane. The amino acid sequence of N-terminals was determined by Edman degradation and performed by a Molecular Biology Resource Facility (University of Oklahoma Health Science Center, Oklahoma City, OK, USA).