Stachyose: an early product of photosynthesis in squash leaves

It was hypothesized that stachyose is translocated by squash because stachyose is supplied to the phloem loading system by the photosynthetic system. To test this hypothesis, ¹⁴CO₂ was supplied to squash leaves. The nonphosphorylated sugars containing ¹⁴C were studied. A large proportion of ¹⁴C appeared in stachyose very early in the time sequence, tending to confirm the hypothesis.     

De Novo Purine Biosynthesis in Intact Cells of Cucurbita pepo

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 [1-¹⁴C]glycine, [2-¹⁴C]glycine, NaH¹⁴CO₃, and H¹⁴COONa 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) NaH¹⁴CO₃ incorporated into ∑Ade (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.

     

Translocation of sugar and tritiated water in squash plants

When ¹⁴C-sugar and THO were simultaneously introduced through a cut side vein or flap of a squash leaf (Cucurbita melopepo, Bailey cv. torticollis) concurrent translocation of ¹⁴C-sugars, T-photosynthates and THO with parallel, almost flat, gradients was observed in the petiole for periods of 1 to 3 hr. Parallel translocation gradients were not observed when ¹⁴C was introduced as ¹⁴CO₂ and T by painting a leaf with THO. Autoradiography of frozen sections to locate the tissues in which THO was moving was unsuccessful. Steam-girdling blocked the movement of ¹⁴C and T when ¹⁴C-glucose and THO were introduced simultaneously by the flap-feeding technique. If THO moved as liquid water in the phloem along with the ¹⁴C-sugars, as blockage by steam girdling suggests, then solution flow of sugar cannot be excluded as a mechanism of translocation.     

Radiosynthesis of 6’-Deoxy-6’[18F]Fluorosucrose via Automated Synthesis and Its Utility to Study In Vivo Sucrose Transport in Maize (Zea mays) Leaves

Sugars produced from photosynthesis in leaves are transported through the phloem tissues within veins and delivered to non-photosynthetic organs, such as roots, stems, flowers, and seeds, to support their growth and/or storage of carbohydrates. However, because the phloem is located internally within the veins, it is difficult to access and to study the dynamics of sugar transport. Radioactive tracers have been extensively used to study vascular transport in plants and have provided great insights into transport dynamics. To better study sucrose partitioning in vivo, a novel radioactive analog of sucrose was synthesized through a completely chemical synthesis route by substituting fluorine-18 (half-life 110 min) at the 6’ position to generate 6’-deoxy-6’[¹⁸F]fluorosucrose (¹⁸FS). This radiotracer was then used to compare sucrose transport between wild-type maize plants and mutant plants lacking the Sucrose transporter1 (Sut1) gene, which has been shown to function in sucrose phloem loading. Our results demonstrate that ¹⁸FS is transported in vivo, with the wild-type plants showing a greater rate of transport down the leaf blade than the sut1 mutant plants. A similar transport pattern was also observed for universally labeled [U-¹⁴C]sucrose ([U-¹⁴C]suc). Our findings support the proposed sucrose phloem loading function of the Sut1 gene in maize, and additionally demonstrate that the ¹⁸FS analog is a valuable, new tool that offers imaging advantages over [U-¹⁴C]suc for studying phloem transport in plants.     

Effect of altered sink: source ratio on photosynthetic metabolism of source leaves

When seven crop species were grown under identical environmental conditions, decreased sink:source ratio led to a decreased photosynthetic rate within 1 to 3 days in Cucumis sativus L., Gossypium hirsutum L., and Raphanus sativus L., but not in Capsicum annuum L., Solanum melongena L., Phaseolus vulgaris L., or Ricinus communis L. The decrease was not associated with stomatal closure. In cotton and cucumber, sink removal led to an increase in starch and sugar content, in glucose 6-phosphate and fructose 6-phosphate pools, and in the proportion of ¹⁴C detected in sugar phosphates and UDPglucose following ¹⁴CO₂ supply. When mannose was supplied to leaf discs to sequester cytoplasmic inorganic phosphate, promotion of starch synthesis, and inhibition of CO₂ fixation, were observed in control discs, but not in discs from treated plants. Phosphate buffer reduced starch synthesis in the latter, but not the former discs. The findings suggest that sink removal led to a decreased ratio inorganic phosphate:phosphorylated compounds. In beans ¹⁴C in sugar phosphates increased following sink removal, but without sucrose accumulation, suggesting tighter feedback control of sugar level. Starch accumulated to higher levels than in the other plants, but CO₂ fixation rate was constant for several days.     

Aspartate Carbamyltransferase : Site of End-Product Inhibition of the Orotate Pathway in Intact Cells of Cucurbita pepo

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 NaH¹⁴CO₃, but not [¹⁴C]carbamylaspartate or [¹⁴C]orotic acid, into uridine nucleotides (ΣUMP). 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 NaH¹⁴CO₃ into ΣUMP by 80% but did not inhibit the incorporation of NaH¹⁴CO₃ 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 NaH¹⁴CO₃ into arginine was stimulated more than 15-fold when both uridine and ornithine were added; (c) incorporation of NaH¹⁴CO₃ 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.     

Spontaneous Phloem bleeding from cryopunctured fruits of a ureide-producing legume

The vasculature of the dorsal suture of cowpea (Vigna unguiculata [L.] Walp) fruits bled a sugar-rich exudate when punctured with a fine needle previously cooled in liquid N₂. Bleeding continued for many days at rates equivalent to 10% of the estimated current sugar intake of the fruit. A phloem origin for the exudate was suggested from its high levels (0.4-0.8 millimoles per milliliter) of sugar (98% of this as sucrose) and its high K⁺ content and high ratio of Mg²⁺ to Ca²⁺. Fruit cryopuncture sap became labeled with ¹⁴C following feeding of [¹⁴C]urea to leaves or adjacent walls of the fruit, of ¹⁴CO₂ to the pod gas space, and of [¹⁴C] asparagine or [¹⁴C]allantoin to leaflets or cut shoots through the xylem. Rates of translocation of ¹⁴C-assimilates from a fed leaf to the puncture site on a subtended fruit were 21 to 38 centimeters per hour. Analysis of ¹⁴C distribution in phloem sap suggested that [¹⁴C]allantoin was metabolized to a greater extent in its passage to the fruit than was [¹⁴C] asparagine. Amino acid:ureide:nitrate ratios (nitrogen weight basis) of NO₃-fed, non-nodulated plants were 20:2:78 in root bleeding xylem sap versus 90:10:0.1 for fruit phloem sap, suggesting that the shoot utilized NO₃-nitrogen to synthesize amino acids prior to phloem transfer of nitrogen to the fruit. Feeding of ¹⁵NO₃ to roots substantiated this conclusion. The amino acid:ureide ratio (nitrogen weight basis) of root xylem sap of symbiotic plants was 23:77 versus 89:11 for corresponding fruit phloem sap indicating intense metabolic transfer of ureide-nitrogen to amino acids by vegetative parts of the plant.     

Sucrose in the free space of translocating maize leaf bundles

Following exposure of portions of mature maize (Zea mays L.) leaf strips to ¹⁴CO₂, xylem exudate from the leaf strips contained [¹⁴C]sucrose. Sucrose was the only sugar in the xylem exudate which was obtained from the cut surface of the leaf strips by reducing the external pressure. The sucrose found in the xylem exudate apparently was obtained from the free space of the vascular bundles, its concentration amounting up to 0.25%. When [¹⁴C]glucose or [¹⁴C]fructose was supplied in the dark to one end of a maize leaf strip, each was taken up by the xylem, and transported to the opposite end. Xylem exudate from such leaf strips contained ¹⁴C-labeled sucrose in addition to the ¹⁴C-labeled hexose. The results of this study support the view that sucrose is loaded into the companion cell-sieve tube complexes from the apoplast of the vascular bundles in the maize leaf.     

Metabolic Requirement of Cucurbita pepo for Boron

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 [³H]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 [¹⁴C]leucine into protein and respiration as measured by O₂ 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%. O₂ 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.     

Bidirectional translocation of sugars in sieve tubes of squash plants

Two streams of sugars moving in opposite directions in the petiole of a half-grown leaf were demonstrated by feeding tritiated glucose to a fully grown leaf of a squash plant (Cucurbita melopepo Bailey) and ¹⁴CO₂ to the half-grown one. Autoradiographic evidence indicates that the movement of both streams occurred within the same sieve tubes. The data do not fit the mass flow theory of translocation which requires unidirectional flow of sugar solution in the lumen of the sieve tube.     

Increased Arginine Biosynthesis during Phosphorus Deficiency : A Response to the Increased Ammonia Content of Leaves

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 < 0.05). Consistent with increased availability of ammonia, the incorporation of NaH¹⁴CO₃ 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 NH₄⁺ for 3 hours resulted in the accumulation of ammonia in the leaves, and a 4-fold increase in the incorporation of NaH¹⁴CO₃ 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.     

Regulation of Pyrimidine Biosynthesis in Intact Cells of Cucurbita pepo

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 [¹⁴C]NaHCO₃, [¹⁴C]carbamylaspartate, and [¹⁴C]orotic acid into uridine nucleotides; (b) the isolation of [¹⁴C]orotic acid when [¹⁴C]NaHCO₃ and [¹⁴C]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.     

Benzyladenine-induced Movement of C-Labeled Photosynthate into Roots of Vitis vinifera

Roots of Vitis vinifera L., were treated with benzyladenine when the plant shoots were 38 cm long. Seventy-two hours after benzyladenine treatment, apical or basal leaves on separate shoots were exposed to ¹⁴CO₂. Control shoots received ¹⁴CO₂ but no benzyladenine. Application of benzyladenine directed ¹⁴C-photosynthate to roots, but a small amount of radioactivity was detected in the shoot tip when ¹⁴CO₂ was administered to an apical leaf. Distribution of radioactivity among the sugar, organic acid, and amino acid fractions was altered by benzyladenine treatment. In all parts of plants with roots treated with benzyladenine and apical leaf fed ¹⁴CO₂, the percentage of the total label in the sugar fraction comprised of fructose was generally more than twice that in control plants.     

Transport and metabolism of adenosine in human blood platelets

The uptake and metabolism of [¹⁴C]- or [³H]adenosine have been studied in suspensions of washed platelets and in platelet rich plasma. The appearance of radio-activity in the platelets and the formation of radioactive adenosine metabolites have been used to determine the uptake. Adenosine is transported into human blood platelets by two different systems: a low K_m system (9.8 μM) which is competitively inhibited by papaverine, and a high K_m system (9.4 mM) which is competitively inhibited by adenine. Adenosine transported via the low K_m system is probably directly incorporated into adenine nucleotides, while adenosine transported through the high K_m system arrives unchanged inside the platelet and is then converted into inosine and hypoxanthine or incorporated into adenine nucleotides.     

Biosynthesis, translocation, and accumulation of betaine in sugar beet and its progenitors in relation to salinity

Like other halophytic chenopods, sugar beet (Beta vulgaris L.) can accumulate high betaine levels in shoots and roots. N,N,N-trimethylglycine impedes sucrose crystallization and so lowers beet quality. The objective of this research was to examine the genetic variability and physiological significance of betaine accumulation in sugar beet and its relatives. Three cultivated genotypes of B. vulgaris and two genotypes of the wild progenitor B. maritima L. were grown with and without gradual salinization (final NaCl concentration = 150 millimolar). At 6 weeks old, all five genotypes had moderately high betaine levels in shoots and roots when unsalinized (averages for all genotypes: shoots = 108 micromoles per gram dry weight; roots = 99 micromoles per gram dry weight). Salinization raised betaine levels of shoots and roots 2- to 3-fold, but did not greatly depress shoot or root growth. The genotype WB-167—an annual B. maritima type—always had approximately 40% lower betaine levels in roots than the other four genotypes, although the betaine levels in the shoots were not atypically low.

The site and pathway of betaine synthesis were investigated in young, salinized sugar beet plants by: (a) supplying 1 micromole [¹⁴C]ethanolamine to young leaf blades or to the taproot sink of intact plants; (b) supplying tracer [¹⁴C]formate to discs of leaf, hypocotyl, and taproot tissues in darkness. Conversion of both ¹⁴C precursors to betaine was active only in leaf tissue. Very little ¹⁴C appeared in the phospholipid phosphatidylcholine before betaine was heavily labeled; this was in marked contrast to the labeling patterns in salinized barley. Phosphorylcholine was a prominent early ¹⁴C metabolite of both [¹⁴C]ethanolamine and [¹⁴C]formate in all tissues of sugar beet. Betaine translocation was examined in young plants of sugar beet and WB-167 by applying tracer [methyl-¹⁴C]betaine to a young expanded leaf and determining the distribution of ¹⁴C after 3 days. In all cases, extensive ¹⁴C translocation to young leaves and taproot sink occurred; neither in the fed leaf nor in sink organs were any ¹⁴C metabolites of betaine detected.

     

Carbon-14 Distribution in Carbohydrates of Immature Zea mays. Kernels Following CO(2) Treatment of Intact Plants

Shortly after Zea mays L. plants were exposed to ¹⁴CO₂, most of the radioactivity in the kernel occurred in the free monosaccharides, glucose and fructose. Later the proportion of ¹⁴C in sucrose increased and that in the monosaccharides declined. These data have been interpreted as showing that the translocated sugar is hydrolyzed prior to or during its movement into the storage cells of the endosperm. This hydrolysis appears to occur in the “pedicel region” of the kernel. After entry into the endosperm tissue, sucrose was rapidly resynthesized from the monosaccharides prior to its utilization in starch synthesis.     

Metabolic fate of 3,4-dichloropropionanilide in plants: the metabolism of the propionic Acid moiety

3,4-Dichloropropionanilide-¹⁴C (propanil) labeled in either the C-1 or C-3 carbon atoms of the propionic acid moiety was applied to the roots of pea (Pisum sativum L.) and rice (Oryza sativa L.) plants in nutrient solution (0.1 mm-0.28 mm). Radioactivity was detected throughout the treated plants, but the greatest labeling was found in the roots. None of the products that contained aniline were radioactive, suggesting that the plants split the propionic acid moiety from propanil. The fate of the propionate moiety of propanil was determined by recovery of ¹⁴CO₂ from plants exposed to propanil-¹⁴C. The time-course of the ¹⁴CO₂ production demonstrated that the intact propionic acid was cleaved from the propanil and subsequently catabolized by the β-oxidation catabolic sequence. The appearance of radioactivity in the shoots was attributed to the incorporation of products of propionate metabolism. Both the susceptible pea plants and the tolerant rice plants converted a high percentage of the administered propanil-¹⁴C to ¹⁴CO₂.     

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (`Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [¹⁴C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of ¹⁴C-label into sink leaves during steady state labeling of a source leaf with ¹⁴CO₂. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[¹⁴C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[¹⁴C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.     

Purine biosynthesis and catabolism in soybean root nodules: incorporation of 14C from 14CO2 into xanthine

Nodulated root systems of soybean plants were exposed to ¹⁴CO₂ in the presence and absence of allopurinol. After 5 h about one-fifth of the label in the perchloric acid-soluble fraction of the nodules was found to be in xanthine in the allopurinol-treated plants. Control plants contained much lower levels of xanthine, but with similar specific activity. Hypoxanthine was not detected in either control or allopurinol-treated plants, even though it would be expected to accumulate in the latter. Degradation of labeled xanthine from allopurinol-treated plants using xanthine oxidase and uricase resulted in the loss of most of the label. The preferential incorporation and accumulation of ¹⁴C from ¹⁴CO₂ into C₆ of xanthine in allopurinol-treated plants is consistent with the involvement of phosphoribosylaminoimidazole carboxylase in the de novo synthesis of purines. The accumulation of xanthine and absence of hypoxanthine in nodules of allopurinol-treated plants confirms earlier observations. In addition, the similar specific activities of ¹⁴C in xanthine in allopurinol-treated and control plants indicate that the xanthine which accumulates in allopurinol-treated plants is the product of de novo purine biosynthesis.     

Metabolism of arginine by aging and 7 day old pumpkin seedlings

The metabolism of arginine by etiolated pumpkin (Cucurbita moschata) seedlings was studied over various time and age intervals by injecting arginine-U-¹⁴C into the cotyledons. At most, 25% of the ¹⁴C was transported from the cotyledon to the axis tissue and the amount of this transport decreased with increasing age of the seedlings. The cotyledons of 25 day old plants contained 60% of the administered ¹⁴C as unmetabolized arginine. Little ¹⁴C was in sugars and it appeared that arginine was the primary translocation product. Time course studies showed that arginine was extensively metabolized and the labeling patterns suggest that different pathways were in operation in the axis and cotyledons. The amount of arginine incorporated into cotyledonary protein show that synthesis and turnover were occurring at rapid rate. Only 25% of the label incorporated into protein by 1.5 hr remained after 96 hr. The label in protein was stable in the axis tissue. By 96 hr 50% of the administered label occurred as ¹⁴CO₂ and it appeared that arginine was metabolized, through glutamate, by the citrio acid cycle in the cotyledons. The experiments showed that an extensive conversion of arginine carbon into other amino acids did not occur.