LONG-TERM DEVELOPMENTAL CHANGES IN XANTHIUM INDUCED BY GIBBERELLIC ACID

One application of gibberellic acid (GA₃) to Xanthium shoots resulted in an initial large stimulation, followed by inhibition, of internode elongation. After presumed translocation of the hormone from the locus of its application to the stem apex several morphological changes were observed. There was a significant increase in number of mitotic figures in the apical meristem and a twofold increase in volume of the apical dome. With time, the rate of leaf production was accelerated about 1.8 times. The phyllotaxis of leaf primordia initiated under the influence of GA:, changed from a (2, 3) contact parastichy pattern in control shoot to a (3, 5) pattern. Final petiole length was smaller than the control, and the absolute rate of lamina expansion decreased under prolonged treatment. Gibberellic acid had a pronounced effect on leaf morphology. GA_a induced the development of lanceolate leaves instead of typical deltoid leaves. The reduction in leaf area coincided with a 32% reduction in the average area of epidermal cells. Plastochron changes were correlated with anatomical and morphological changes during the course of leaf development.     

REGULATORY ROLE OF INDOLE-3-ACETIC ACID AND GIBBERELLIC ACID IN VEGETATIVE DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

A single application of gibberellic acid to young internodes significantly accelerated the rate of internode growth and the rate of leaf production in shoots of Xanthium pennsylvanicum Wallr. The average duration of one plastochron in treated plants was reduced to 43% of control levels. Gibberellic acid also had a pronounced morphogenetic effect on leaves so that the area and leaf length of treated plants were both significantly reduced. Depending upon concentration, auxin had both inhibitory and promotive effects on Xanthium shoots. Indole-3-acetic acid markedly altered the response of the gibberellic acid-treated internodes and those located above and below the site of application. In addition, high auxin concentrations induced the formation of adventitious roots in treated internodes. Auxin also brought about significant reductions in the length and area of leaves developed under the influence of this hormone.     

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.     

PHYLLOTACTIC CHANGE INDUCED BY GIBBERELLIC ACID IN XANTHIUM SHOOT APICES

Gibberellic acid (GA) treatment of vegetative shoots of Xanthium leads to a change in phyllotaxis as diagnosed in transverse sections of apical buds. A method of analysis is proposed for estimating the phyllotactic parameters, the plastochron ratio, a, and the divergence angle, α, from measurements of the angular and radial positions of leaf primordia in sections. GA treatment significantly decreases the plastochron ratio, a, from 1.35 in controls, to 1.19 in GA-treated plants, as shown by an analysis of variance, but has no significant effect on the divergence angle. The estimates of a and α are compared with the parameters of theoretical phyllotaxis models, leading to the designation (2, 3) for controls, and (3, 5) for GA-treated plants, where the integers 2, 3, and 5 designate sets of contact parastichies. The change in a is interpreted as indicating a change in the relative position at which leaf primordia are initiated in the apical meristem, and this effect is discussed in relation to theories of leaf initiation.     

ACTIVITY OF MARGINAL AND PLATE MERISTEMS DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

The mitotic and biosynthetic activities of the marginal and plate meristems were studied during the entire course of leaf development of Xanthium pennsylvanicum. In contrast to statements in the literature, marginal meristem activity is long in duration, as assayed by the mitotic counts and H³-thymidine incorporation. This me istem is active 23 days. The plate meristem is active for an additional 3 days after cessation of cell division in the marginal meristem, but the total duration of its mitotic activity is also approximately 23 days. Numerous periclinal cell divisions of the plate meristem form additional cell layers and contribute to the growth of the lamina in thickness. Incorporation of H³-thymidine increased during the course of leaf development. Cells between plastochronic ages 0 and 2.0 incorporated more of the radioisotopic precursor than those of younger leaf primordia. The uptake and incorporation of H³-thymidine into nuclear DNA was more sluggish during the early stages of development than in the more expanded leaves. No DNA synthesis was demonstrated after cessation of cell division in the leaf lamina. Metabolic or endomitotic DNA synthesis after leaf plastochron index (LPI) 3.0 seems improbable. No significant differences in the incorporation of H³-thymidine could be demonstrated between the marginal and plate meristems. This would indicate no distinct biosynthetic differences between the two meristems. The definitions of the marginal and plate meristems of Xanthium leaves were formulated in view of the above findings.     

CHANGES IN PROTEIN BIOSYNTHESIS DURING GIBBERELLIC ACID INDUCED INDUCTION AND FORMATION OF ANTHERIDIUM IN THE FERN ANEMIA PHYLLITIDIS

Changes in protein biosynthesis have been studied during the induction and formation of antheridium in Anemia phyllitidis .
Based on incorporation of ¹⁴C-amino acid mixture into TCA precipitable material two distinct phases of accelerated protein biosynthesis were observed. First phase initiates at 4th day, while the second at 8th day of development. The first phase is likely associated with antheridium induction and second with spermatogenesis. From electrophoretic pattern of proteins on stained acrylamid gels and from radioactivity profiles of labelled proteins distinct quantitative differences between vegetative and reproductive prothalli were observed at different stages of antheridial development. Radioactivity profiles reveal characteristic pattern of each stage of antheridial differentiation.     

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.     

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.     

THE EFFECT OF GIBBERELLIC ACID ON LEAF AREA AND DRY-MATTER PRODUCTION IN MAJESTIC POTATO

When gibberellic acid (50 p.p.m. in aqueous solution) was sprayed twice or six times at weekly intervals on potato plants (var. Majestic) with a low or high nitrogen supply it did not affect rate of leaf production on the main axis, but caused earlier senescence of leaves, especially with the more frequent spraying, and inhibited leaf production and growth on laterals of the high-nitrogen plants at nodes 10 and 11 but not at other nodes. This central region of the stem appears to have a low growth potential, probably because it lies midway between two zones of active growth, viz. the basal branches and the younger leaves on the main stem. Competition between these is increased by gibberellic acid. Gibberellic acid increased leaf area even when lack of nitrogen was restricting growth but this did not produce extra dry matter. Tuber weight was increased more in high-nitrogen plants by two sprayings than by six sprayings. The net assimilation rate of low-nitrogen plants was halved by spraying but was not changed in high-nitrogen plants where the value was similar to that of low-nitrogen control plants. The high-nitrogen plants had absorbed nearly all the available nitrogen between the second and third harvests, but plants treated with gibberellic acid, nevertheless, had more total dry weight and tuber dry weight than the controls. The nitrogen content of the leaves expressed on an area basis was lower in sprayed plants and, with continued spraying, fell at the third harvest to equal that of low-nitrogen plants. Evidently, the effect of gibberellic acid depended on the interaction between the rate of application and the nitrogen supply, but further work is necessary to define the conditions that give the maximal effect on dry-matter production.     

INTERNODE ELONGATION IN XANTHIUM PLANTS TREATED WITH GIBBERELLIC ACID PRESENTED IN TERMS OF RELATIVE ELEMENTAL RATES

Xanthium plants were grown vegetatively and their developmental stages were designated by a previously described plastochron index (PI). Internodes of plants, both treated with gibberellic acid (GA₃) and untreated, were marked with India ink and photographed during 3 successive days. The relative elemental rates of elongation d(dX/dt)/dX were estimated between 15.7 and 19.0 plastochrons. The rate of growth of the GA₃-treated internodes was at least twice that of the control. The emerging pattern of acropetal internode elongation was similar in both GA₃-treated and control plants. Only rates of growth were significantly higher in the GA₃-treated plants. The acropetal pattern of internode elongation was the opposite of the basipetal pattern observed in Xanthium leaves but followed the acropetal pattern observed in Helianthus and Phaseolus internode growth.     

CONTROL OF INTERCALARY GROWTH IN THE SCAPE OF GERBERA BY AUXIN AND GIBBERELLIC ACID

With the inflorescence removed, intercalary growth can be maintained in the scape of Gerbera jamesonii by application of gibberellic acid (GA, gibberellin A₃) or indole-3-acetic acid (IAA); the latter usually promotes more rapid and greater elongation than the former because of a greater effect on older tissues. Simultaneous application of the two substances, even when both are at optimal levels, promotes more rapid elongation than either substance alone; in fact, the rate of elongation may equal that of the intact scape. In decapitated scapes (receptacle and involucral bracts removed with the inflorescence), GA and IAA promote cell elongation with reduced or no cell division. In deflowered scapes (receptacle and involucral bracts intact) both GA and IAA promote cell division, as well as cell elongation, so that the pattern of scape elongation is nearly the same as that for intact scapes. Apparently the bracts and receptacle contribute something required for cell division which acts in concert with GA and IAA. Deflowered and decapitated scapes elongate at nearly the same rates initially; thus the rate of elongation does not depend on cell division. The ultimate length of the scape is dependent on cell number and, hence, cell division, since deflowered scapes attain greater lengths than those that are decapitated.     

REGULATION OF GROWTH AND CELLULAR DIFFERENTIATION IN DEVELOPING AVENA INTERNODES BY GIBBERELLIC ACID AND INDOLE-3-ACETIC ACID

Auxin (IAA) at physiological concentrations causes significant reduction of GA₃-promoted growth in excised Avena stem segments. IAA is thus considered to be a gibberellin antagonist in this system. It was found to act non-competitively in repressing GA₃-augmented growth in these segments. In intercalary meristem cells at the base of the elongating internode, GA₃ blocks cell division activity and causes a marked increase in cell lengthening. IAA substantially promotes lateral expansion in comparable intercalary meristem cells, particularly in the vicinity of vascular bundles underlying the epidermis. It also alters the plane of cell division in differentiating stomata. IAA at high concentrations (10⁻³, 10⁻⁴ m ), in combination with GA₃, overrides the effects of GA₃ on cell lengthening, while with low concentrations of IAA (10⁻⁹, 10⁻¹⁰m ), the effects of GA₃ are clearly dominant. At intermediate concentrations of IAA (10⁻⁶, 10⁻⁷m ), in the presence of GA₃, the effects of this treatment on cell differentiation closely parallel the pattern of differentiation in untreated tissue. It is postulated that a lateral gradient of auxin and gibberellin could control cell expansion in long epidermal cells during intercalary growth of the internode.     

CELL DIVISION AND CELL ELONGATION IN LEAF DEVELOPMENT OF XANTHIUM PENSYLVANICUM

Maksymowych, Roman. (Villanova U., Villanova, Pa.) Cell division and cell elongation in leaf development of Xanthium pensylvanicum. Amer. Jour. Bot. 50(9) : 891–901. Illus. 1963.—Cell division in different parts of the lamina and cell enlargement of the upper epidermis and palisade mesophyll were studied in vertical and horizontal planes during the entire period of growth. The leaf plastochron index (L.P.I.) was used for designation of developmental stages of the leaf. From cell-length data and measurements of cell area the absolute rates of elongation (dX/dpl) and relative rates of elongation (dlnX/dpl) were calculated. The increase in number of cells in the early plastochrons is exponential and cell division stops at about L.P.I. 3.0. Divisions cease first at the tip and last in the basal lobes of the leaf, indicating a basipetal trend of this process. Cells are elongating while division is in progress, though this elongation proceeds at low rates and for a limited time. Palisade cells elongate in the vertical plane at higher rates and at least 1 plastochron sooner than the upper epidermis. The latter cells, however, expand in area with higher absolute and relative rates, and about 2 plastochrons in advance of the palisade mesophyll. The rates are not constant during the whole period of development but are represented by the bell-shaped curves with maximal peaks around L.P.I. 3.0 for the middle portion of the lamina. The increase in volume of the 2 types of cells stops around L.P.I. 5.0, or shortly after. In addition to unequal durations of cellular enlargement, both tissues expand at differential rates, which for the upper epidermis is high in the horizontal plane but low in the vertical plane, while the opposite is true for the palisade mesophyll. It is suggested that palisades and spongy mesophyll are separated and intercellular spaces formed during the course of development because of the greater rate of expansion in area of the upper epidermis.     

QUANTITATIVE ANALYSIS OF LEAF DEVELOPMENT IN XANTHIUM PENSYLVANICUM

Maksymowych , Roman . (Villanova U., Villanova, Pa.) Quantitative analysis of leaf development in Xanthium pensylvanicum. Amer. Jour. Bot. 46(9): 635–644. Illus. 1959.—An attempt was made to find a quantitative way of describing the development of the leaf and to correlate the developmental processes, designating precisely their sequence. The processes were presented in terms of the absolute and relative rates of leaf length, expansion of lamina in surface, increase in thickness, rates of cell division of leaf 9 and 13, and tissue differentiation of 3 portions of the lamina. All rates were estimated over the entire period of development, from initiation of a primordium to its maturity. The leaf plastochron index (L.P.I.) was used as a morphological time-scale. The relative plastochron rates were used for the purpose of correlation of the developmental processes. Leaf 9 elongates exponentially up to 3.0 L.P.I. with an average relative rate (dlnL/dpl) of about 0.78 pl^-1, and it stops growing around 8.0 L.P.I. The lamina stops elongating about 1.5 plastochrons before the petiole. The tip of the lamina expands its surface at a constantly lower relative rate than the middle and the basal portions of the blade. The average relative rate of expansion in area (dlnA/dpl) for the whole lamina is 1.7pl^-1 during the exponential stage. Differentiation of the laminar tissues proceeds basipetally, from the tip toward the base of the leaf. The relative rate of expansion of lamina in thickness (dlnT/dpl) is 0.55 pl^-1 at 1.5 L.P.I. and after 4.0 L.P.I. all cells cease elongating in a plane perpendicular to the leaf surface. The formation of cells proceeds exponentially up to 3.0 L.P.I. and about this time cell divisions stop in all parts of the lamina. The mean relative rate of cell formation (dlnC/dpl) at the exponential phase is 1.41 pl^-1, an increase of about 31% per day. At least 27 generations of cells are involved in the process of leaf formation and the generation time was calculated to be 0.5 plastochron or 2.2 days.     

A MORPHOGENETIC STUDY OF LEAF ABSCISSION IN PHASEOLUS

Leaf abscission in Phaseolus vulgaris L., the red kidney bean, is presumed to involve a precisely regulated set of interlocking reactions, all of which are subject to control and many of which are detectable morphologically and histochemically. Four sequential stages which culminate in separation are distinct in the laminar abscission region. These include 1) pith cell breakdown, which may not be related to abscission; 2) cell division; 3) cellular differentiation; and 4) cortical and vascular cell breakdown. Correlation of the time and the nature of these structural changes with the period of effectiveness of certain applied growth regulators provides some interesting insights into the nature of the regulatory mechanisms in abscission. A reevaluation of some contemporary concepts of abscission, particularly the characterization of abscission as a senescence phenomenon, is suggested.