INCORPORATION OF H3-THYMIDINE IN LEAF NUCLEI OF XANTHIUM PENNSYLVANICUM

The basic kinetics and the pattern of incorporation of H³-thymidine was studied in the leaf lamina of Xanthium pennsylvanicum. A method of foliar absorption was used to incorporate the radioisotope into leaf nuclei. The autoradiographic techniques employed provided data on the amount of the isotope incorporated. It was determined that 10 μc/ml (sp. act. 6.7 c/mmole) of H³-thymidine with 1–8 hr of isotopic growth and 4 hr of postisotopic growth gave the most satisfactory results. The percent of labelled nuclei and the number of grains per nucleus were presented as functions of isotopic and postisotopic growth periods. Distribution of grains in the nuclei approximated the Poisson distribution at 1 hr of isotopic growth. Increased time of isotopic growth changed the pattern of grain distribution. No deleterious effects were observed using an 8-hr period of isotopic growth, but prolonged incubation time significantly decreased the proportion of mitotic figures in the lamina. The amount of incorporation of the DNA precursor expressed as percent of labelled nuclei was linear to about 16 hr of isotopic growth and thereafter decreased gradually. As indicated by the average number of grains per nucleus, H³-thymidine incorporation increased to about 16 hr, and soon after reached a saturation level. The percent of labelled nuclei and the number of grains per nucleus decreased as a function of the postisotopic growth period. However, they were significantly greater in the lamina near the vein than in the lamina region at some distance from the vein. The radioactive precursor was initially absorbed by the cells of the lamina and was subsequently translocated into the vascular system. There it was circulated and made available to the dividing cells near veins of the lamina. This region may be a metabolically distinct part of the lamina with significantly higher rates of incorporation and mitotic turnover.     

DNA SYNTHESIS,CELL DIVISION,AND CELL DIFFERENTIATION DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

Leaf growth consists of two basic processes, cell division and cell enlargement. DNA synthesis is an integral part of cell division and can be studied with autoradiographic techniques and incorporation of some labeled precursor. Studies were made on the synthesis of nuclear DNA through incorporation of ³H-thymidine in various parts of the lamina during the entire course of leaf development of Xanthium pennsylvanicum. The time course analysis of DNA synthesis was correlated with cell division and rates of cell enlargement. Significant differences in ³H-thymidine incorporation were found in various parts of the lamina. Cell division and DNA synthesis were highest in the early stages of development. Since no ³H-thymidine was incorporated after cessation of cell division (LPI 2.8) in the leaf lamina, it appears that DNA synthesis is not needed for enlargement and differentiation of Xanthium cells. Rates of cell enlargement were negligible in the early development and reached their maximum after cessation of mitoses, between plastochron ages (LPI) 3 and 4. Cells matured between LPI's 5 and 6. Enzymatic activity was correlated with cell division and cell differentiation at various stages of leaf development.     

LEAF DEVELOPMENT IN SENECIO ROWLEYANUS (COMPOSITAE)

Prolonged apical growth of the leaf primordium and the presence of distinct marginal meristems do not occur in Senecio rowleyanus. Intercalary cell divisions accompanied by radial expansion of derivatives from an adaxial meristem account for the spherical shape of the leaf. The “window” in the lamina marks the position of the adaxial meristem and precludes interpretation of the leaf as being unifacial. Stomata are mesoperigenous and anomocytic in type. Schizogenous secretory canals occur in both the leaf and the stem, and their association with vascular bundles is discussed. The anatomy of the leaf is interpreted in terms of xeromorphy.     

DEVELOPMENT OF THE LAMINA IN XANTHIUM ITALICUM REPRESENTED BY THE PLASTOCHRON INDEX

Maksymowych , Roman (Villanova U., Villanova, Pa.), and Ralph O. Erickson . Development of the lamina in Xanthium italicum represented by the plastochron index. Amer. Jour. Bot. 47(6):51–459. Illus. 1960.—Xanthium plants were grown vegetatively, and leaves, whose developmental age was specified by a previously described leaf plastochron index (L.P.I.), were measured and studied histologically. Leaf initiation occurs before L.P.I.–6.0. Initiation of the lamina occurs at about L.P.I. –4.8, when the primordium is about 0.22 mm. long. Mitotic divisions in the marginal and plate meristems continue until about L.P.I. 1.7, when the leaf is about 37 mm. long, a period of about 23 days. Between L.P.I. 1.0 and 3.0, approximately, there is a rapid 4-fold increase in thickness of the lamina, with development of the characteristic palisade and spongy mesophyll tissues. Virtually no further increase in thickness occurs after L.P.I. 4.0, but the lamina continues to increase in length until about L.P.I. 6.0, when it has reached a mature length of about 180 mm. A statistical analysis has been made of planes of division in the marginal meristem, which in some respects fails to support published histogenetic schemes for this meristem.     

THE EARLY ACTION OF THE FLORAL STIMULUS ON MITOTIC ACTIVITY AND DNA SYNTHESIS IN THE APICAL MERISTEM OF XANTHIUM STRUMARIUM

Vegetative plants of Xanthium strumarium (a short-day species) were induced to flower by exposure to a single 16-hr long night. By cutting off the induced leaf (half-expanded leaf) at various times, it was established that, by 8 hr after the end of the long night, a sufficient amount of floral stimulus had reached the meristem to induce a flowering response. The following sequence of events occurred in both the peripheral and central zones of the apical meristem of induced plants: 1) a rise in the mitotic index beginning at 28 hr after the end of the long night and culminating at 36 and 56 hr; 2) a stimulation of DNA synthesis starting at 32–36 hr and reaching a maximum at 60 hr; 3) an increase in nucleolus diameter starting at 32 hr. The cell population in the meristems of both vegetative and induced plants displayed a similar distribution, with about 80 % of the nuclei with the 2C amount of DNA. The comparison of the kinetic data concerning the mitotic index and DNA synthesis indicated that one of the early effects of the floral stimulus in the peripheral and central zones was the release in mitosis of cells whose nuclei were in the postsynthetic (G₂) phase of the mitotic cycle. In the pith-rib meristem, the following events were recorded: 1) a stimulation of DNA synthesis starting at 20 hr; 2) a rise of the mitotic index beginning at 28 hr; 3) the vacuolation and elongation of cells starting at 48 hr. All these events occurred well before the initiation of bract and flower primordia, which began at 96 and 136 hr, respectively. Neither stimulation of mitotic activity nor flowering occurred in the meristems of plants subjected to a long night interrupted at its midpoint by a 5-min light break. The results are discussed in relation to the early events which are known to occur in the meristems of other photoperiodic species in transition to flowering.     

DISTRIBUTION OF 3H-THYMIDINE IN ARABIDOPSIS VEGETATIVE MERISTEMS AFTER 5-IODODEOXYURIDINE TREATMENT

Arabidopsis thaliana vegetative meristems, growing under short photoperiods, respond to the application of IUdR by precocious floral morphogenesis. ³H-thymidine was used to label cells active in DNA synthesis and study the effect of analogue application on the amount and distribution of DNA synthesis throughout the meristem during 78 hr subsequent to IUdR treatment. Photomicrographs, autoradiographs, and composite plots of label distribution in transparent superimposed sequential sections revealed a non-uniform distribution of labelling in control vegetative meristems, which typically contained a central and axial core incorporating little ³H-thymidine and a peripheral flanking tunica region which contained densely labelled cells. The ratio of labelled cells in the peripheral region to labelled cells in the central region was about 10:1 in the controls. In meristems pretreated with IUdR there was a brief suppression of ³H-thymidine incorporation during 6–12 hr after treatment, followed by two waves of enhanced incorporation in the peripheral region, and a progressive increase in the frequency of labelled cells in the central core of the meristem. After 78 hr the frequency of labelled cells in the central core of IUdR-treated meristems was 8-fold higher than in untreated meristems, and the frequency in the peripheral regions was about the same in both IUdR and control series. The enhancement in amount and uniformity of DNA synthesis after temporary inhibition by IUdR parallels the normal enhancement which is observed when vegetative meristems are transferred to long photoperiods causing floral induction.     

COMPARATIVE MORPHOGENESIS OF THE DIMORPHIC LEAVES OF CYAMOPSIS TETRAGONOLOBA

Cyamopsis tetragonoloba, guar, produces two leaf forms: a simple leaf and a trifoliate leaf. The steps in the development of each of these forms have been investigated in an attempt to determine the precise point at which the leaf primordium becomes destined to produce one or the other characteristic leaf shapes. Up to 140 μ in length the leaf primordia are morphologically indistinguishable. If a simple leaf is to be formed the marginal meristem remains continuous and initiates only lamina. If a trifoliate leaf is to be formed the continuity of the marginal meristem is interrupted by a group of “pocketal” cells dividing it into an upper lamina meristem and a basal leaflet buttress.     

THE FERTILE SPIKE OF OPHIOGLOSSUM PETIOLATUM. I. MECHANISM OF ELONGATION

The frond of Ophioglossum consists of a sterile segment and a fertile segment or spike. An investigation of fertile spike elongation reveals that growth of the spike proceeds by activity of an intercalary meristem located in the most distal region of the peduncle subtending the sporangial area. Anatomical comparisons of all regions of developing spikes, counts of mitotic figures along the length of spikes of various ages, determination of cell lengths of peduncle ground parenchyma cells, and historadioautography of spikes treated with H³-thymidine confirms the presence in the apical portion of the peduncle of a region of frequently dividing cells intercalated between two regions of more mature tissues. Marking experiments indicate that the petiole of the sterile segment of the frond elongates in a similar fashion. Although this type of intercalary meristem is rather common in angiosperm flower scapes and peduncles, this is the first detailed analysis of this type of growth in a pteridophyte genus.     

SYNTHETIC CAPACITIES OF CHROMOSOME FRAGMENTS CORRELATED WITH THEIR ABILITY TO MAINTAIN NUCLEOLAR MATERIAL

Onion (Allium cepa) and bean (Vicia faba) root tip cells containing many micronuclei, derived from x-ray-induced chromosome fragments, were exposed to H³-thymidine and H³-cytidine to determine the ability of such fragments to undergo DNA and RNA synthesis. Only a few micronuclei in onion and many in bean roots synthesize nucleic acid simultaneously with their main nuclei. A few micronuclei labeled with H³-thymidine undergo mitotic chromosome condensation along with the main nuclei, while the unlabeled ones never do so. The onset of nucleic acid synthesis as well as mitosis in micronuclei appears to be under generalized cellular control. Although all chromosomes and chromosome fragments at telophase give a positive reaction for a silver stainable nucleolar fraction, in the subsequent interphase only some micronuclei, derived from such chromosome fragments, are found to maintain nucleoli; others lose them with time. Those micronuclei which maintain nucleoli, perhaps due to the presence of specific chromosomal regions, are also active in DNA and RNA synthesis. These results are compatible with the concept that nucleoli and associated chromosome regions play an important role in the primary biosynthetic processes of the cell.     

INCORPORATION OF H3-THYMIDINE INTO SHOOT AND ROOT APICES OF CERATOPTERIS THALICTROIDES

Gifford , Ernest M., Jr . (U. California, Davis.) Incorporation of H³-thymidine into shoot and root apices of Ceratopteris thalictroides. Amer. Jour. Bot 47(10): 834–837. Illus. 1960.—The localization of tritiated thymidine in apical meristems of Ceratopteris thalictroides by the autoradiographic method is described. Intact, floating plants of the fern were placed in 1/2 strength Hoagland's inorganic nutrient solution containing H³-thymidine (10 μc/ml.) for 3 days. The material was killed, dehydrated and embedded in paraffin. Autoradiographic stripping film (AR 10 Kodak) was applied to serial sections. After an appropriate exposure period, the film was developed and the sections with the superimposed film were stained lightly with Harris' hematoxylin. The autoradiographs revealed the presence of the H³-thymidine in nuclei of the large, individualized apical cells of shoots and roots which is proof of DNA synthesis. In no instances were these nuclei unlabeled. If endomitotic reduplication is excluded the results of these studies lend support to the concept that apical cells actually do divide and perhaps at a higher rate than envisioned by other workers. Considerable cytoplasmic labeling occurred and its significance to general problems of DNA synthesis is discussed.     

MORPHOLOGY OF MARSILEA VESTITA. I. ONTOGENY AND MORPHOLOGY OF THE SUBMERGED AND LAND FORMS OF THE JUVENILE LEAVES

During their ontogeny, the primordia of the juvenile leaves of Marsilea plants in sterile culture develop 1, 2 or 4 marginal meristems, and these, in turn, contribute cells to the young leaf by anti- and periclinal cell divisions. The final leaves are unifid, bifid, or quadrifid, depending on how many marginal meristems develop, and this is determined early in the ontogeny of the leaf. The mechanism which determines whether or not a marginal meristem develops may fluctuate, as shown by the existence of trifid leaves. Two forms of juvenile leaves are produced, those in a liquid medium, which in many respects resemble the adult quadrifid submerged leaves, and those on a solid medium, which in many respects resemble the adult land leaves.     

INCORPORATION OF TRITIATED THYMIDINE DURING VARIOUS STAGES OF LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

³H-thymidine was incorporated into leaf tissue of Xanthium pennsylvanicum during the stage of active cell division, during cellular differentiation, and into mature cells. Incorporation into nuclear DNA was high in the early stages of development. No nuclear incorporation was found after cessation of cell division. However, significant incorporation could be demonstrated in cytoplasm of differentiating and mature cells. Depending upon the time of growth in the radioisotope and the time of growth after treatment, ³H-thymidine, or its metabolized fraction, was incorporated into the secondary wall depositions of epidermal cells, mesophyll parenchyma cells, xylem cells, and chloroplasts. Autoradiographic technique and liquid scintillation spectrometry were used in these studies. The significance of ³H-thymidine incorporation into various organelles is discussed in relation to cell metabolism and its regulation during leaf development.     

Developmental anatomy of foliage leaves,bracts, calyx and corolla inPharbitis nil

The structure and ontogeny of the foliage leaves, bracts, bracteoles, calyx and corolla ofPharbitis nil were investigated, with special reference to the development of the lamina and the procambium. Reproductive organs used are those of a terminal inflorescence and axillary flowers induced by a single 16 hr dark period given to the seedling. The foliage leaf consists of the petiole and the broad lamina. Bracts show various forms and structures, which fluctuate from a lower leafy bract to an upper scaly one in a terminal inflorescence. The sepal is scaly. The corolla is funnel-shaped, and composed of five wedge-shaped petals. In the lamina of the foliage leaf primordium, marginal growth is followed by active growth by the plate meristem, and procambial strands of lateral veins differentiate from the residual meristem. The primordium of the lowest bract of the terminal inflorescence has already been initiated before the dark period, and develops into the bract, the residual meristem disappearing after the treatment. The leafy bract shows marginal growth and growth by the plate meristem similar to that of the foliage leaf, but of short duration. The activity of marginal growth of the scaly bract and the sepal decreases rapidly and procambial strands of lateral veins differentiate acropetally from highly vacuolated cells. The activity of marginal growth of the petal decreases gradually, and derivatives of the marginal meristem divide as a plate meristem. The corolla tube is initiated by co-operation of interprimordial growth and marginal growth of petal primordia.     

CYTOKININ- AND GIBBERELLIC ACID-INDUCED EFFECTS ON THE STRUCTURE AND METABOLISM OF SHOOT APICAL MERISTEMS IN OPUNTIA POLYACANTHA (CACTACEAE)

The dormant axillary buds of Opuntia polyacantha can be activated by either cytokinins or gibberellic acid. Under the influence of benzylaminopurine (BAP), the axillary bud meristem increases greatly in size and becomes mitotically active. The primordia produced by the meristem develop as normal photosynthetic leaves. Gibberellic acid (GA) also causes the meristem to become mitotically active, but the meristem does not increase in size. The primordia produced under the influence of GA develop as normal cactus spines. Leaf-producing meristems and spine-producing meristems have the same zonation, despite the differences in size. The meristems are composed of a uniseriate tunica, a central mother cell zone, peripheral zone, and a pith rib meristem. The mitotic activity of each of the zones in the leaf-producing meristem differs significantly from the mitotic activity of the corresponding zones in the spine-producing meristem.     

Morphogenetic Studies on the Distribution and Activities of Leaf Meristems in Ferns

In very young sporophytes of Dryopteris and Osmunda, the leafprimordia originate very close to the shoot apical cell andshow early differentiation of an apical cell, rapid growth,and an early transition from distal to marginal growth. In successively older primordia of adult Dryopteris, a gradualelaboration in the size of the leaf apical cell takes placeand the greatest size is attained before lateral pinnae beginto be formed. With the formation of lamina, the apical cellgradually decreases in size and is transformed into the marginaltype of meri-stematic cell, when the leaf unrolls. In ferns with a homogeneous marginal meristem, which consistsof a uniform layer of cells with an equal capacity for growth,a simple, entire leaf is formed, e.g. Phyllitis and Platyceriumand where an initially homogeneous marginal meristem becomesheterogeneous, with a consequential differentiation of areasof unequal growth, a lobed or pinnate configuration, as in Blechnumand Lomnaria, or a compound leaf, as in Dryopteris, results. There are some indications of the inception of vascular elementsbeing due to the activity of functioning meristems, the processbeing a basipetal one.     

QUANTITATIVE STUDIES OF PINNULE DEVELOPMENT IN THE FERNS ADIANTUM RADDIANUM AND CHEILANTHES VIRIDIS

Pinnule development was investigated in two fern species, Adiantum raddianum Presl cv. Decorum and Cheilanthes viridis (Forsk.) Swartz, by using clearings to facilitate the recording of mitotic divisions. Both species were found to possess a marginal meristem. This meristem consists of both a marginal row of large initials and a submarginal meristematic zone. The marginal meristem in these ferns is responsible for establishing the layers of the lamina, providing new cells which by enlargement will expand the pinnule, establishing general pinnule form, initiating the procambial stands, and forming the false indusia. The cells of the submarginal meristem were found to divide parallel to the pinnule margin more frequently if they were to become ground tissue, while dividing perpendicular to the margin more frequently if they were to become procambial. Details of vein dichotomies were also studied. Perimeter expansion was found to be associated with dichotomy of the veins, and venation pattern was found to be correlated with leaf form. The marginal meristem is active from the time of pinnule initiation until the pinnule reaches about 50% of its final length or width. Leaf development in leptosporangiate ferns resembles the traditional concept of development in angiosperms somewhat more than it does the more recent concepts. It is clear, though, that there is not a high degree of convergence in the marginal growth of fern and angiosperm leaves.     

DNA synthesis in the giant salivary chromosomes of Drosophila virilis prior to pupation

Both two-wavelength microspectrophotometry of Feulgen-stained whole nuclei and autoradiography of H³-thymidine incorporation by giant salivary chromosomes in Drosophila virilis demonstrate a net decrease in the relative rate of salivary DNA synthesis during the late third instar and prepupal stages of development. Amounts of DNA-Feulgen per nucleus were distributed into several classes, the means of which closely approximated values projected as geometric multiples of the basic somatic DNA level estimated from hemocyte nuclei of the same larvae. Comparison of DNA polytene class frequencies showed no statistical difference between male larvae of different development stages, although female prepupae showed a greater frequency of nuclei in higher polytene classes when compared to male prepupae of the same age. Comparison of chromosomal H³-thymidine incorporation with previously described H³-histidine incorporation suggests that the amino acid labeling, which reaches a maximum during the prepupal period, has a physiological significance distinct from chromosomal endoreplication.     

Macromolecule synthesis leading to cell division in Tetrahymena pyriformis after replacement of required amino acids

Summary Tetrahymena pyriformis W were brought to a nonmultiplying state by removal of required amino acids from their growth medium. After amino-acid replacement, the incorporation rates of H³-uridine, H³-thymidine and H³-leucine were measured by the autoradiographic method. Following amino-acid replacement, the first response was detected in RNA synthesis, then protein synthesis, then DNA synthesis and, lastly, in cell division. Amino-acid deprived cells showed a 23% net increase in DNA content, a result supporting the view of others that protein synthesis is not necessary for the initiation of DNA synthesis but is necessary for the maintainance of DNA synthesis.     

EVIDENCE FOR CYTOPLASMIC DNA IN ROOT CELLS OF NICOTIANA

Sterile root cultures from Nicotiana tabacum were grown with H³-thymidine added to the medium for various intervals. Incorporation of the labeled nucleoside into nuclear DNA occurred in a fraction of the nuclei which increased with time. In addition, the cytoplasm of all cells incorporated enough tritium to be readily detected by autoradiography. The tritium was not removed by hydrolysis in 1 N HCl at 60°C for 10 minutes, but was removed by digestion in a DNase solution which also removed nuclear DNA. The amount of tritium in the cytoplasm increased during the first 2 hours, but did not appear to increase significantly during the following 5 hours. If the roots were transferred to unlabeled medium after 2 hours, the label was diluted faster than expected by growth without turnover of the labeled component. If FUdR was added to the unlabeled medium, the depletion occurred faster during the first 6 hours, but later appeared to level off so that at 10 hours these cultures did not differ from those incubated without FUdR. However, the addition of an excess of unlabeled carrier had no effect on the rate of depletion of the cytoplasmic label. Actinomycin D, which inhibited the incorporation of H³-cytidine into RNA in the root tips, had no effect on the incorporation of H³-thymidine into the cytoplasmic component. However, Mitomycin C or a high concentration of deoxyadenosine inhibited the incorporation of H³-thymidine into the cytoplasmic component as well as into the nuclear DNA. It is concluded that H³-thymidine is incorporated into a cytoplasmic fraction which has the characteristics of DNA, with a measurable rate of turnover. This fraction is synthesized regardless of whether or not the nucleus is synthesizing DNA. Although the function of cytoplasmic fraction is not yet known, it does not appear to be that of supplying precursors for the synthesis of the nuclear DNA.