Nitrogen-containing Compounds in the Shoot System of Pisum arvense L. I. Amino Compounds in the Bleeding Saps from Petioles

A technique is described for collecting bleeding saps from thepetioles of defoliated plants of the field pea. Analytical studiesand ¹⁴CO₂-feeding experiments indicate that these saps are physicallyrelated to the fluids delivered to the xylem by the nodulatedroot. There is a progressive decrease in the concentration ofamino-nitrogen in the bleeding saps recovered from the successivepetioles up a stem. This decrease is manifest at any time ofthe day or night and is evident in all ages of plants. It issuggested that it results from the accumulation of a proportionof the incoming amino-acids and amides in living tissues ofthe stem. As the stem ages, it appears to become less effectivein abstracting materials from the xylem. Stem tissues oftenshow a more intensive uptake of asparagine than of other constituentssuch as aspartic acid, glutamine, and homoserine. The abovestudies suggest that much of the nitrogen coming from the rootis immediately sequestered by the lower half of the shoot. Asimilar effect is observed if ¹⁴Clabelled bleeding sap is fedto complete, unlabelled shoots via the transpiration stream.     

Photosynthesizing Leaves and Nodulated Roots as Donors of Carbon to Protein of the Shoot of the Field Pea (Pisum arvense L.)

In Pisum arvense, the amides and amino-acids normally suppliedto the shoot in the transpiration stream transfer carbon toprotein largely throught the amino-acids, aspartic acid (+asparagine),glutarnic acid (+glutamine), threonine, lysine, arginine, andproline. Carbon from carbon dioxide enters the protein of photosynthesizingtissues through an essentially complementary set of amino-acidsincluding glycine, alanine, serine, valine, and the aromaticamino-acids tyrosine, phenylalnine, and histidine. Young tissuesof the shoot synthesize certain amino-acids de novo by metabolismof sugars supplied from photosynthesizing leaves. Each mature leaf on a shoot contributes carbon to current synthesisof protein at the shoot apex. Sucrose accounts for more than90 per cent of the labelled carbon leaving any age of leaf whichhas been fed with ¹⁴CO₂. Upper leaves supply labelled assimilatesdirectly to the shoot apex, and the radiocarbon from these assimilatesis subsequently incorporated into a wide range of amino-acidunits of protein. The majority of the labelled assimilates exportedfrom a lower leaf move downwards to the root and nodules and,in consequence, the amino-acids and amides associated with rootmetabolism are strongly represented among the compounds eventuallylabelled in the apical region of the shoot.     

The localization of enzymes in the cotyledons of Pisum arvense L. during germination

Summary Enzyme activity is not uniformly distributed through the cotyledon of Pisum arvense. Initially the peripheral region, certain scattered cells of the storage tissue and the procambium show a high level of activity of succinic dehydrogenase, cytochrome oxidase, acid phosphatase and esterase. Activity of acid phosphatase declines sharply after the first day of germination; activity of the other enzymes declines after about three days. In the storage tissue, where activity is lower initially, it declines after about five days and is correlated with the disappearance of the reserves. The pattern of alkaline phosphatase activity is similar except that activity is lower in the procambium but increases in the sieve-elements during differentiation of the phloem. 5-nucleotidase and glucose-6-phosphatase activity is low throughout the cotyledon but it also increases to a significant level in the sieve-elements. Activity of starch synthesizing enzymes is high in the parenchymatous bundle sheath, where they may be involved in the pathway from lipids to soluble carbohydrates.     

Nuclear Changes in the Cotyledons of Pisum arvense L. during Germination

In the cells of the cotyledons of Pisum arvense L. there isa close correlation between cell volume, nuclear volume, andnuclear DNA level. The cells of the epidermis are at the 2Clevel of DNA, those of the hypodermus vary from 2C to 4C, whilethose of the storage tissues range from 4C to 16C. During germinationthe nucler increase in size In the storage tissues there isa three to fourfold increase, which is accompanied by an increasein lobing of the nucler Simultaneously the DNA level of thenucler decreases by about 50 per cent. There are parallel changesin nuclear histone levels. Initially nuclear RNA level is lowbut in any given cell it increases to a maximum at the timethe storage reserves of the cell begin to disappear, after whichit declines. The level of cytoplasmic RNA is high initiallyin the tissues at the abaxial side of the cotyledon. Duringthe first few days of germination it declines until the over-alllevel is uniformly low, after which there is a further smalldecline.     

Movement of Assimilated Nitrogen from the Root System of the Field Pea (Pisum arvense L.)

Changes in the composition of the amino fraction of bleedingsap collected from decapitated root systems of field pea werestudied during the period of transition from cotyledon nutritionto the commencement of nitrogen fixation by root nodules. Outputand composition of bleeding sap are described for uninoculatedseedlings following application to the rooting medium of nitrate,ammonium, or urea. Assimilation by the root system of any externalsource of nitrogen results in progressive increase in the outputof all bleeding-sap components, particularly the amide asparagine.The relative proportions of the various amino compounds andthe balance of organic and inorganic nitrogen in the bleedingsap were found to vary with the source of nitrogen made availableto the root system.     

Determination of Stelar Elements in Roots of Pisum sativum L.

Cytochemical studies of esterase activity in 0.5 mm segmentsfrom root tips of Pisum sativum explanted for up to 9 days inbasal culture medium containing 2 per cent sucrose showed retentionof this activity. During this time, all segments from the secondand third 0.5 mm segments of the root tip developed xylem elementsas did the proximal end of the first segment. No xylem elementswere found in the 12–14 cells behind the quiescent centre.It is concluded that the central group of meristem cells aregenerally programmed to form tissues of the stele immediatelyon leaving the quiescent centre, and that the programming forxylem and phloem elements occurs as a second step. Pisum sativum L., garden pea, determination, histochemistry, esterases, stele, root     

The development of polysomes in the seed of Pisum arvense

1. Ribosomal preparations from dormant seed, cotyledon and growing tissue from Pisum arvense were examined. 2. Polysomes were obtained from growing tissues under all conditions used. 3. Such particles were obtained from seed immediately after imbibition only when 5mm-zinc sulphate was included in the medium used for extraction. 4. No polysomes were obtained from dry seed. 5. Extracts of dry seed showed limited incorporation of phenylalanine into protein. 6. Extracts of seed after imbibition showed enhanced activity in incorporation of phenylalanine amounting to 72% of the activity found in extracts of growing tissue. 7. A nuclear fraction from dry seed was able to incorporate ATP into acid-precipitable material. 8. It is concluded that the protein-synthesizing system of the dry seed is limited by the low concentration of functional polysomes.     

Uptake of Inorganic Phosphate by Plastids Purified from the Roots of Pisum sativum L.

Pea root plastids are able to take up inorganic phosphate ina time-dependent reaction. The rate of uptake is reduced inthe presence of triose phosphates, though the kinetics of uptakedo not appear to conform to a simple Michaelis-Menten interpretation.Inorganic phosphate taken up by such preparations is lost fromthe plastids upon addition of triose phosphates. Unlike chloroplasts,root plastids do not appear to accumulate inorganic phosphateabove the concentration in the surrounding medium. Key words: Pisum, plastid, phosphate uptake     

Nucleic Acid, Protein, and Starch Synthesis in Developing Cotyledons of Pisum arvense L.

During the cell-division period of cotyledon development inPisum arvense L. cell volume increases slightly but nuclearvolume shows little variation and the DNA content remains atthe 2C to 4C level. During the main period of cell expansionthere is a close correlation between cell volume, nuclear volume,and nuclear DNA content, the nuclei of the largest storage cellsfinally attaining the 64C level. The rate of RNA synthesis increasesseveral days after the increase in DNA has begun and at thesame time accumulation of reserve protein and starch begins.RNA and starch synthesis apparently cease some time before maturationbut protein synthesis continues until the seeds are ripe. Cotyledondevelopment was found to comprise two distinct phases: an initialphase of cell division and differentiation during which DNA,RNA, and protein per unit volume of cell decline; and a phaseof reserve accumulation in which DNA per unit volume of cellremains constant but RNA and protein per unit volume increase,starch synthesis is initiated, and all the cotyledon cells assumethe properties of storage cells.     

The Inhibition of Chemiluminescence in Pisum Sativum L. Roots by Certain Metal Ions

The effect of rare metal ions on the activity of the peroxidase system in Pisum sativum L. roots was studied by luminol-dependent chemiluminescence. Trivalent ions of scandium, gallium, indium, and lanthanum, to different extents, inhibit the chemiluminescence of damaged P. sativum roots. A decreased generation of superoxide due to the formation of the complex between metal ions and NADP can underlie the inhibited activity of peroxidase system. The possible mechanism of inhibition of the peroxidase system activity by metal ions is discussed.     

Potassium Transport in Corn Roots : II. The Significance of the Root Periphery

The relative transport capabilities of the cells of the root periphery and cortex were investigated using a variety of experimental techniques. Brief (30 seconds to 1 minute) exposures with the penetrating sulfhydryl reagent, N-ethyl maleimide (NEM), and the impermeant reagent, p-chloromercuribenzene sulfonic acid (PCMBS), dramatically reduced ⁸⁶Rb⁺ (0.2 millimolar RbCl) uptake into 2 centimeter corn (Zea mays [A632 × (C3640 × Oh43)]) root segments. Autoradiographic localization studies with [³H]NEM and [²⁰³Hg]PCMBS demonstrated that, during short term exposures with either reagent, sulfhydryl binding occurred almost exclusively in the cells of the root periphery.

Corn root cortical protoplasts were isolated, and exhibited significant K⁺(⁸⁶Rb⁺) influx. The kinetics for K⁺ uptake were studied; the influx isotherms were smooth, nonsaturating curves that approached linearity at higher K⁺(Rb⁺) concentrations (above 1 millimolar K⁺). These kinetics were identical in shape to the complex kinetics previously observed for K⁺ uptake in corn roots (Kochian, Lucas 1982 Plant Physiol 70: 1723-1731), and could be resolved into a saturable and a first order kinetic component.

The existence of a hypodermal apoplastic barrier was investigated. The apoplastic, cell wall binding dye, Calcofluor White M2R, appeared to be excluded from the cortex by the hypodermis. However, experiments with damaged roots indicated that this result may be an artifact resulting from the binding of dye to the epidermal cell walls. Furthermore, [²⁰³Hg] PCMBS autoradiography demonstrated that the hypodermis was not a barrier to apoplastic movement of PCMBS.

These results suggest that although cortical cells possess the capacity to absorb ions, K⁺ influx at low concentrations is limited to the root periphery. Cortical cell uptake appears to be repressed under these conditions. At higher concentrations, cortical cells may function to absorb K⁺. Such a model may involve regulation of cortical cell ion transport capacity.

     

The degradation of ribonucleic acid in the cotyledons of Pisum arvense

1. A ribonuclease has been partially purified from the cotyledons of germinating seed of Pisum arvense. 2. The enzyme degrades ribopolynucleotides to adenosine 3'-phosphate, guanosine 3'-phosphate and the cyclic nucleotides cytidine 2',3'-phosphate and uridine 2',3'-phosphate; no resistant ;core' remains. 3. The activity of RNA-degrading enzymes in the cotyledons increases to a maximum during the first 5 days of germination, passes through a minimum around the eighth day, and thereafter increases again. 4. Ion-exchange chromatography of methanol-soluble extracts of cotyledons revealed the presence, amongst other components, of the 2'-, 3'- and 5'-phosphates of cytidine and uridine, the 3'- and 5'-phosphates of adenosine, and guanosine 5'-phosphate. 5. Seed soaked in a solution containing [(32)P]orthophosphate gave a methanol-soluble fraction containing labelled nucleoside 5'-phosphates, but nucleoside 2'- and 3'-phosphates were not labelled. 6. It is believed that the nucleoside 2'- and 3'-phosphates arise by the action of ribonuclease on cotyledon RNA.     

Three New Phenolic Amides from the Roots of Eggplant (Solanum melongena L.)

The isolation of four phenolic amides, four phenolic compounds and an aromatic amine from the roots of eggplant is described. The phenolic amides were identified as N-trans-feruloyl tyramine (V), N-trans-p-coumaroyl tyramine (VII), N-trans-feruloyl octopamine (VIII) and N-trans-p-coymaroyl octopamine (IX). The three amides V, VIII and IX are new compounds. Furthermore, four phenolic compounds were identified as vanillin (I), isoscopoletin (II), ethyl caffeate (IV) and ferulic acid (VI). The aromatic amine was identified as p-aminobenzal-dehyde (III).     

The Kinetics of Phloem Loading of Valine in the Shoot of a Nodulated Legume (Lupinus albus L. cv. Ultra)

Phloem loading of several amino acids (D- and L-Val, Arg, Asn,Asp, Leu) was studied in shoots of L. albus using a phloem bleedingtechnique on both intact plants and detached shoots fed viathe transpiration stream. Val was singled out for intensivestudy due to the minimal amount of metabolism it underwent inthe shoot For the amino acids studied, the relationship between xylem,phloem, and leaflet concentrations was determined by the interactionof rates of xylem supply, metabolism, and export. At elevatedxylem fluid concentrations, low rates of loading of D-Val intothe phloem and little metabolism in the tissues resulted inhigh levels in the leaflets. For other amino acids (Arg, Asp,Leu) rapid metabolism in the leaflets prevented a build-up inconcentration in either phloem or leaflets. Asn was rapidlytransferred to the phloem, thus high levels in the xylem leadto high concentrations in the phloem without greatly affectingleaflet concentrations. L-Val responded in a manner intermediatebetween Asn and D-Val. A detailed study of L-Val showed it to be loaded into the phloemagainst a concentration gradient in both stem and leaflets.Some of this Val originated from the transpiration stream atboth locations but in the leaflets as much as 64% of the Valoriginated from other sources, e.g. recent photosynthesis. L-Valsupplied to the phloem in the stem was derived from a largestorage pool and did not come directly from the xylem fluid.As a consequence the rate of stem loading was independent ofshort-term fluctuations in the xylem fluid Val concentration.L-Val entering the leaflets in the xylem initially bypassedthe large storage pool and was loaded directly into the phloem.However, after 350 min the pools had reached an equilibriumand rate of phloem loading was dependent on total leaflet concentration.     

The Distribution of {beta}-Glycerophosphatase in Young Roots of Pisum sativum L.

The distribution of ß-glycerophosphatase activityin young roots of Pisum sativum, cultivar Alaska, has been examinedby biochemical and histochemical methods. Results obtained bythe two approaches are broadly similar, and indicate that highenzyme activity is associated with cells of the root cap, outerlayers of the cortex, differentiating xylem elements and phloemfibres, and cortical cells surrounding emerging lateral roots.The significance of this distribution in relation to a possiblefunction of ß-glycerophosphatase is discussed.     

Carbon and Nitrogen Transfer from Vegetative Organs to Ripening Seeds of Field Pea (Pisum arvense L.)

The fate of the carbon from photosynthetically assimilated ¹⁴CO₂and the nitrogen of rootapplied ¹⁵NO₃ was studied by harvestingplants at intervals after a week's feeding period, either early(34–41 d after sowing) during vegetative growth, or late(112–19 d) as plants were fruiting. Only 23 per cent of the early fed ¹⁴C but more than 90 per centof the early-fed ¹⁵N survived in plants until maturity (155d). Efficiency of transfer of early-fed ¹⁴C to seeds was 2 percent, that for early-fed ¹⁵N, 51 per cent. Assimilatory activitybefore flowering had little direct relevance to the carbon nutritionof seeds but provided approximately one-fifth of the seeds'requirement for nitrogen. Early or late-fed ¹⁵N was mobilized slowly for reproductivedevelopment, seeds at all 15 reproductive nodes deriving benefit,late-forming ones more so than those developing earlier. Vegetative organs on a young shoot lost previously acquired¹⁵N at rates within the range 0.5–2.4 per cent per day.The released nitrogen was first taken up by the root and youngerparts of the shoot, but much was later traced to the seeds. Late feeding of ¹⁴C or ¹⁵N resulted in a high (74–6 percent) efficiency of transfer to seeds, most of this appearingto be donated by, or cycled through, vegetative parts of thereproductive zone of the shoot. The significance of past and current assimilation in fruit nutritionis discussed.     

Identification of Shoot Apical Meristem Cells Committed to Form Vascular Elements in Pisum sativum L. and Vicia faba L.

A cytochemical study of naphthol AS-D esterases in vegetativeshoot apices of Pisum sativum and Vicia faba L. has shown thepresence of carboxyl esterases (E.C. 3.1.1.1 [EC] .) in those meristemcells already committed to form vascular elements. These cellsform a sequence linking the morphologically identifiable procambiumto the cells of the tunica layers at a site either already identifiableas the next primordium or which will form the next primordium.The implications of this result are briefly discussed in relationto the control of primordia formation and procambial cell development. Pisum sativum, Vicia faba, determination, vascular tissue, shoot apex, cytochemistry     

Biochemical and Physiological Changes During Maturation of Fruit of the Field Pea (Pisum arvense L.)

Quantitative changes in the carbohydrates, soluble nitrogenouscompounds, and specific proteins of seeds and pods of fieldpea have been followed during two seasons and related to theoverall pattern of growth of the embryo and other parts of thefruit. Reserves of solutes are established in the pod and inthe seed coat and endosperm of the seed some time before theembryo commences its exponential phase of growth. Later, thesereserves disappear and it is estimated that their mobilizationmight provide, at the most, only one-fifth of the embryo's requirementsfor carbon and nitrogen. Starch and certain albumin-type proteins accumulate relativelyearly in the life of the embryo. Globulin-type proteins, bycontrast, are laid down only after the cotyledons are almostfull size and after most of the reserve of starch has been accumulated.The content of hemicellulose, the principal carbohydrate ofthe embryo, increases steadily as the embryo grows. Neitherhemicellulose nor globulin is found in appreciable amounts inorgans other than the cotyledons. Each organ of the fruit has its own highly characteristic poolof free amino acids. Orderly and reproducible changes occurin the composition of these pools during fruit development,suggesting that variations in the amounts of specific compoundsmay have special significance in the programme of developmentand synthesis in the fruit.