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The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions.

In Coffea arabica leaves, the purine ring of theobromine (3,7-dimethylxanthine) and caffeine (1,3,7-trimethylxanthine) is provided by de novo purine biosynthesis: (a) [14C]glycine, [14C]bicarbonate, and [14C]formate were incorporated into inosine 5[prime]- monophosphate (IMP), sum of adenine nucleotides ([sigma]Ade), theobromine, and caffeine; and (b) incorporation of [14C]formate into IMP, [sigma]Ade, theobromine, and caffeine was inhibited by azaserine, a known inhibitor of de novo purine biosynthesis. Capacity of coffee leaves to salvage added purines was demonstrated by incorporation of [14C]hypoxanthine into [sigma]Ade and the incorporation of [14C]adenosine, [14C]adenine, [14C]inosine, and [14C]hypoxanthine into both theobromine and caffeine. Consistent with synthesis of theobromine from two separate purine nucleotide pools, one synthesized de novo and one via salvage, added xanthine 5[prime]-monophosphate (XMP), inosine, or hypoxanthine failed to reduce the incorporation of [14C]formate into theobromine but diluted the specific radioactivity of [14C]adenosine and [14C]adenine incorporated into theobromine. Evidence that theobromine is not the immediate precursor of caffeine is provided: (a) [14C]xanthine was incorporated into caffeine but not into theobromine; (b) exogenous xanthine diluted the specific radioactivity of caffeine synthesized from [14C]adenine and [14C]hypoxanthine but caused accumulation of radiolabel in theobromine; (c) allopurinol, a known inhibitor of the conversion of hypoxanthine to xanthine, reduced incorporation of [14C]adenine and [14C]hypoxanthine into caffeine but caused accumulation of radiolabel in theobromine; and (d) incorporation of [14C]formate into caffeine, but not into theobromine, was reduced by added XMP, inosine, or hypoxanthine.

Young, fully expanded leaves from 7-month-old P-deficient citrus rootstock seedlings had levels of nonprotein arginine that were 10- to 50-fold greater than those from P-sufficient control plants. Arginine content of the protein fraction increased 2- to 4-fold in P-deficient leaves. Total arginine content, which averaged 72 ± 6 micromoles per gram dry weight of P-sufficient leaf tissue (mean ± se, n = the four rootstocks) was 207, 308, 241, and 178 micromoles in P-deficient leaves from Citrus limon cv rough lemon, Poncirus trifoliata × C. sinensis cv Carrizo citrange and cv Troyer citrange, and P. trifoliata cv Australian trifoliate orange, respectively. For each rootstock, the accumulation of arginine paralleled an increase in the activity of the pathway for the de novo biosynthesis of arginine. The ratio of the nanomoles NaH14CO3 incorporated into the combined pool of arginine plus urea per gram fresh weight intact leaf tissue during a 3-hour labeling period for P-deficient to P-sufficient plants was 91:34, 49:11, 35:11, and 52:41, respectively. When P-deficient plants were supplied with P, incorporation of NaH14CO3 into arginine plus urea was reduced to the level observed for the P-sufficient control plants of the same age and arginine ceased to accumulate. Arginase and arginine decarboxylase activity were either unaffected or slightly increased during phosphorus deficiency. Taken together, these results provide strong evidence that arginine accumulation during phosphorus deficiency is due to increased activity of the de novo arginine biosynthetic pathway.

The accumulation of arginine in leaves of four citrus rootstock cultivars during P deficiency has been demonstrated to be due to increased de novo synthesis rather than decreased catabolism or increased protein degradation (E Rabe, CJ Lovatt, 1984, Plant Physiol 76: 747-752). In this report, we provide evidence (a) that the increased activity of the arginine biosynthetic pathway observed for citrus rootstocks grown under P-deficient conditions for 7 months is due to an increase in the concentration of ammonia in leaves of P-deficient plants and (b) that ammonia accumulation and removal through arginine systhesis are early responses to phosphorus deficiency for both a woody perennial, rough lemon ( Citrus limon ), and an herbaceous annual, summer squash ( Cucurbita pepo ). Transferring 5-day-old squash plants to a phosphorus-deficient nutrient solution for only 10 days resulted in a 2-fold increase in the concentration of nitrate in the youngest fully expanded leaves (YFE). Concomitantly, the specific activity of nitrate reductase doubled and the ammonia content of P-deficient YFE leaves increased to a concentration significantly greater that of leaves from healthy control plants (P 14 CO 3 into arginine plus urea doubled during phosphorus deficiency and arginine accumulated. Despite the accumulation of nitrate and ammonia in YFE leaves during phosphorus deficiency, the total nitrogen content of these leaves was less than that of the healthy control plants. Similar results were obtained for rough lemon. Nitrate content of the YFE leaves increased 1.5- and 3.0-fold in plants deprived of phosphorus for 6 and 12 weeks, respectively. Ammonia content of the leaves increased as P deficiency progressed to 1.4 ± 0.08 mg (± se, n = 4) per gram dry weight, a level 1.8-fold greater than that of the P-sufficient control plants. During P deficiency de novo arginine biosynthesis in rough lemon increased 10-fold. Immersing the petiole of YFE leaves from P-sufficient squash and rough lemon plants in 50 millimolar NH 4 + for 3 hours resulted in the accumulation of ammonia in the leaves, and a 4-fold increase in the incorporation of NaH 14 CO 3 into arginine plus urea. Taken together, these results provide strong evidence that the accumulation of nitrate and ammonia in leaves is an early response of both woody and herbaceous plants to P deprivation. The data are consistent with the hypothesis that increased de novo arginine biosynthesis in leaves during P deficiency is in response to ammonia content of the leaves.

Lovatt et al. (1979 Plant Physiol 64: 562-569) have previously demonstrated that end-product inhibition functions as a mechanism regulating the activity of the orotic acid pathway in intact cells of roots excised from 2-day-old squash plants (Cucurbita pepo L. cv Early Prolific Straightneck). Uridine (0.5 millimolar final concentration) or one of its metabolites inhibited the incorporation of NaH(14)CO(3), but not [(14)C]carbamylaspartate or [(14)C]orotic acid, into uridine nucleotides (SigmaUMP). Thus, regulation of de novo pyrimidine biosynthesis was demonstrated to occur at one or both of the first two reactions of the orotic acid pathway, those catalyzed by carbamylphosphate synthetase (CPSase) and aspartate carbamyltransferase (ACTase). The results of the present study provide evidence that ACTase alone is the site of feedback control by added uridine or one of its metabolites. Evidence demonstrating regulation of the orotic acid pathway by end-product inhibition at ACTase, but not at CPSase, includes the following observations: (a) addition of uridine (0.5 millimolar final concentration) inhibited the incorporation of NaH(14)CO(3) into SigmaUMP by 80% but did not inhibit the incorporation of NaH(14)CO(3) into arginine; (b) inhibition of the orotate pathway by added uridine was not reversed by supplying exogenous ornithine (5 millimolar final concentration), while the incorporation of NaH(14)CO(3) into arginine was stimulated more than 15-fold when both uridine and ornithine were added; (c) incorporation of NaH(14)CO(3) into arginine increased, with or without added ornithine when the de novo pyrimidine pathway was inhibited by added uridine; and (d) in assays employing cell-free extracts prepared from 2-day-old squash roots, the activity of ACTase, but not CPSase, was inhibited by added pyrimidine nucleotides.

The occurrence of the complete orotic acid pathway for the biosynthesis de novo of pyrimidine nucleotides was demonstrated in the intact cells of roots excised from summer squash ( Cucurbita pepo L. cv. Early Prolific Straightneck). Evidence that the biosynthesis of pyrimidine nucleotides proceeds via the orotate pathway in C. pepo included: ( a ) demonstration of the incorporation of [ 14 C]NaHCO 3 , [ 14 C]carbamylaspartate, and [ 14 C]orotic acid into uridine nucleotides; ( b ) the isolation of [ 14 C]orotic acid when [ 14 C]NaHCO 3 and [ 14 C]carbamylaspartate were used as precursors; ( c ) the observation that 6-azauridine, a known inhibitor of the pathway, blocked the incorporation of early precursors into uridine nucleotides while causing a concomitant accumulation of orotic acid; and ( d ) demonstration of the activities of the component enzymes of the orotate pathway in assays employing cell-free extracts. Regulation of the activity of the orotate pathway by end product inhibition was demonstrated in the intact cells of excised roots by measuring the influence of added pyrimidine nucleosides on the incorporation of [ 14 C]NaHCO 3 into uridine nucleotides. The addition of either uridine or cytidine inhibited the incorporation of [ 14 C]NaHCO 3 into uridine nucleotides by about 80%. The observed inhibition was demonstrated to be readily reversible upon transfer of the roots to a nucleoside-free medium. Experiments employing various radiolabeled precursors indicated that one or both of the first two enzymes in the orotate pathway are the only site(s) of regulation of physiological importance.

Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [(3)H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron-deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [(14)C]leucine into protein and respiration as measured by O(2) consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O(2) consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems.

Previous work has provided evidence that plants may require boron to maintain adequate levels of pyrimidine nucleotides, suggesting that the state of boron deficiency may actually be one of pyrimidine starvation. Since the availability of pyrimidine nucleotides is influenced by their rates of synthesis, salvage, and catabolism, we compared these activities in the terminal 3 centimeters of roots excised from boron-deficient and -sufficient squash plants (Cucurbita pepo L.). Transferring 5-day-old squash plants to a boron-deficient nutrient solution resulted in cessation of root elongation within 18 hours. However, withholding boron for up to 30 hours did not result in either impaired de novo pyrimidine biosynthesis or a change in the sensitivity of the de novo pathway to regulation by end product inhibition. Boron deprivation had no significant effect on pyrimidine salvage or catabolism. These results provide evidence that boron-deficient plants are not starved for uridine nucleotides collectively. Whether a particular pyrimidine nucleotide or derivative is limiting during boron deprivation remains to be examined.

The capacity of intact cells of roots excised from summer squash plants (Cucurbita pepo L. cv Early Prolific Straightneck) to synthesize purine nucleotides de novo was investigated. Evidence that purine nucleotides are synthesized de novo included: (a) demonstration of the incorporation of [1-(14)C]glycine, [2-(14)C]glycine, NaH(14)CO(3), and H(14)COONa into total adenine nucleotides; (b) observation that the addition of azaserine or aminopterin, known inhibitors of de novo purine synthesis in other organisms, blocked the incorporation of these precursors into adenine nucleotides; and (c) demonstration that the purine ring synthesized from these precursors was labeled in a manner consistent with the pathway for de novo purine biosynthesis found in microorganisms and animal tissues. Under optimal conditions, the activity of this pathway in roots excised from 2-day-old squash plants was 244 +/- 13 nanomoles (mean +/- standard error, n = 17) NaH(14)CO(3) incorporated into summation operatorAde (the sum of the adenine nucleotides, nucleoside and free base) per gram tissue during the 3-hour incubation period.The possible occurrence of alternative enzymic reactions for the first steps of de novo purine biosynthesis was also investigated. No conclusive evidence was obtained to support the operation of alternative enzymic reactions in the intact cell of C. pepo.