Leaf‐shape remodeling: programmed cell death in fistular leaves of Allium fistulosum

Some species of Allium in Liliaceae have fistular leaves. The fistular lamina of Allium fistulosum undergoes a process from solid to hollow during development. The aims were to reveal the process of fistular leaf formation involved in programmed cell death (PCD) and to compare the cytological events in the execution of cell death to those in the unusual leaf perforations or plant aerenchyma formation. In this study, light and transmission electron microscopy were used to characterize the development of fistular leaves and cytological events. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) assays and gel electrophoresis were used to determine nuclear DNA cleavage during the PCD. The cavity arises in the leaf blade by degradation of specialized cells, the designated pre‐cavity cells, in the center of the leaves. Nuclei of cells within the pre‐cavity site become TUNEL‐positive, indicating that DNA cleavage is an early event. Gel electrophoresis revealed that DNA internucleosomal cleavage occurred resulting in a characteristic DNA ladder. Ultrastructural analysis of cells at the different stages showed disrupted vacuoles, misshapen nuclei with condensed chromatin, degraded cytoplasm and organelles and emergence of secondary vacuoles. The cell walls degraded last, and residue of degraded cell walls aggregated together. These results revealed that PCD plays a critical role in the development of A. fistulosum fistular leaves. The continuous cavity in A. fistulosum leaves resemble the aerenchyma in the pith of some gramineous plants to improve gas exchange.     

Hypoxic stress triggers a programmed cell death pathway to induce vascular cavity formation in Pisum sativum roots

Flooding at warm temperatures induces hypoxic stress in Pisum sativum seedling roots. In response, some undifferentiated cells in the primary root vascular cylinder start degenerating and form a longitudinal vascular cavity. Changes in cellular morphology and cell wall ultrastructure detected previously in the late stages of cavity formation suggest possible involvement of programmed cell death (PCD). In this study, cytological events occurring in the early stages of cavity formation were investigated. Systematic DNA fragmentation, a feature of many PCD pathways, was detected in the cavity‐forming roots after 3 h of flooding in situ by terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling assay and in isolated total DNA by gel electrophoresis. High molecular weight DNA fragments of about 20–30 kb were detected by pulse‐field gel electrophoresis, but no low‐molecular weight internucleosomal DNA fragments were detected by conventional gel electrophoresis. Release of mitochondrial cytochrome c protein into the cytosol, an integral part of mitochondria‐dependent PCD pathways, was detected in the cavity‐forming roots within 2 h of flooding by fluorescence microscopy of immunolabeled cytochrome c in situ and in isolated mitochondrial and cytosolic protein fractions by western blotting. DNA fragmentation and cytochrome c release remained confined to the undifferentiated cells in center of the root vascular cylinders, even after 24 h of flooding, while outer vascular cylinder cells and cortical cells maintained cellular integrity and normal activity. These findings confirm that hypoxia‐induced vascular cavity formation in P. sativum roots involves PCD, and provides a chronological model of cytological events involved in this rare and understudied PCD system.     

In situ stems: preservation states and growth habits of the Pennsylvanian (Carboniferous) calamitaleans based upon new studies of Calamites Sternberg, 1820 in the Duckmantian at Brymbo,North Wales,UK

Calamitalean stems that are preserved in growth position in Carboniferous sedimentary deposits have been described many times in the literature as either pith casts or stem casts. Many of these in situ stems show branching, which gives some information on their patterns of growth. Their manner of preservation is discussed in the light of a new stand of well‐preserved in situ stem and branch pith casts of Calamites discovered in mid to upper Duckmantian sandstone at Brymbo in the Wrexham Coalfield of North Wales. Analysis of a brown mineralized layer surrounding the casts and below the black compression remains of the stem tissues has shown the presence of goethite, muscovite, quartz and kaolin. Deposition of these minerals around the inside of the central stem cavity would have provided rigidity and sufficient support, while the pith cavity filled with sediments. The outer tissues would then have been compressed to form a thin coal layer around the mineralized infill of the pith cavity. Cross sections of stems were found clustered together in relatively small areas, and kernel density map and nearest neighbour analysis suggest that each small patch of these pith casts represents an individual plant spread by rhizomatous growth. Stems found in ironstone nodules are external casts of leafy stems preserved by the deposition of siderite on their surfaces. A length of rhizome found at Brymbo was similarly preserved as a cast in ironstone.     

The predominant role of the pith in the Growth and development of internodes in Liquidambar styraciflua (Hamamelidaceae). I. Histological basis of compressive and tensile stresses in developing primary tissues

Quantitative changes in cell pattern in the pith, cortex, cortical collenchyma, and epidermis were followed in developing internodes of Liquidambar to examine the cellular basis of compressive and tensile stresses in organized shoot growth. Initially, the highest rates of cell multiplication occur in the pith, followed successively by the epidermis, cortex, and cortical collenchyma. As internodes enter the phase of maximum elongation growth, mitotic activity begins to shift acropetally, accompanied by pronounced changes in cell pattern. The highest rates of cell multiplication now occur in the pith and cortex and continue until the cessation of internode growth. Concomitantly, reduced rates of cell division in peripheral tissues result in rapid increases in rates of cell elongation in the cortical collenchyma and epidermis. Attention is focused on the role of continued cell division in developing internodes with emphasis on differences in rates of cell multiplication between inner and outer tissues affecting patterns of tissue stress. For example, rapid and sustained increases in cell number in the pith, accompanied by growth of readily extensible pith cells, result in the development of compressive forces driving the growth of internodes. Conversely, continuing divisions in less extensible collenchyma and epidermal cells can relieve threshold tensile stresses resulting from the continuous stretching of these tissues by the developing pith. The concept that the passive extension of peripheral tissues, especially the epidermis, control the rate of internode elongation is viewed as an oversimplification of the interacting role of compressive and tensile forces in organized growth and development.     

Shaping the shoot: the relative contribution of cell number and cell shape to variations in internode length between parent and hybrid apple trees

Genetic control of plant size and shape is a promising perspective,particularly in fruit trees, in order to select desirable genotypes.A recent study on architectural traits in an apple progeny showedthat internode length was a highly heritable character. However,few studies have been devoted to internode cellular patterningin dicotyledonous stems, and the interplay between the two elementarycell processes that contribute to their length, i.e. cell divisionand elongation, is not fully understood. The present study aimedat unravelling their contributions in the genetic variationof internode length in a selection of F₁ and parent genotypesof apple tree, by exploring the number of cells and cell shapewithin mature internodes belonging to the main axes. The resultshighlighted that both the variables were homogeneous in samplescollected either along a sagital line or along the pith width,and suggest that cell lengthening was homogeneous during internodedevelopment. They allowed the total number of cells to be estimatedon the internode scale and opened up new perspectives for simplifyingtissue sampling procedures for further investigations. Differencesin internode length were observed between the genotypes, inparticular between the parents, and partly resulted from a compensationbetween cell number and cell length. However, genetic variationsin internode length primarily involved the number of cells,while cell length was more secondary. These results argue foran interplay between cellular and organismal control of internodeshape that may involve the rib meristem. Key words: Elongation, growth, histogenesis, Malusxdomestica Borkh, pith Received 2 January 2008; Revised 22 January 2008 Accepted 29 January 2008     

DEVELOPMENT OF CHAMBERED PITH IN STEMS OF PHYTOLACCA AMERICANA L. (PHYTOLACCACEAE)

An integrated microscopic (light and electron microscopy) and macroscopic investigation of chambered pith development was made of Phytolacca americana L. Terminal internodes have a solid pith cylinder in contrast to the alternating diaphragms and chambers occurring in subjacent pith. Macroscopically, chambers and diaphragms of any one internode are of equal size. Microscopically, diaphragms vary in height within an internode (from 1–6 cells high). Nevertheless, all diaphragms become thicker circumferentially (5–12 cells high) and connect with long files of intact peripheral pith cells. Diaphragm cells have a large centrally positioned vacuole with a thin, parietal layer of cytoplasm; nuclei, mitochondria, endoplasmic reticulum, and unidentified organelles differentiate in the cytoplasm of diaphragm cells. Although schizogenous activity has most often been implicated as the mechanism by which chambered pith develops in vegetative organs of angiosperms, the results of this study show that cavities in pokeweed result from both schizogenous and lysigenous mechanisms. Schizogeny is suggested by the fact that central pith cells of terminal internodes are longer and thinner walled than peripheral pith cells arranged in vertical files, thus indicating elongation of cells as a possible result of internode elongation. The precise developmental pattern and arrangement of chambers and diaphragms also suggest schizogenous processes. Lysigenous or enzymatic activity is indicated by the fact that cavities are bounded by broken cells, and wall fragments and organelles are often found within enlarging cavities. Chamber formation occurs continuously acropetally and centrifugally in the central pith. A comparison of diaphragms is made with Liriodendron tulipifera and Juglans nigra in an attempt to resolve differences in structure and terminology regarding the differentiation of chambered and diaphragmed pith.     

Sucrose Mobilisation in Sugarcane Stalk Induced by Heterotrophic Axillary Bud Growth

A theoretical high-yield sugarcane biofactory can be idealised as containing culm tissue that functions as a secondary source tissue rather than a sink. To investigate this potential process, heterotrophic axillary bud outgrowth from sugarcane (Saccharum spp. hybrids) setts was used as a model system to demonstrate that sucrose is a mobilisable carbon source. The outgrowth and subsequent biomass accumulation of axillary buds from two-eye setts of mature sugarcane stalks grown in the dark was used to measure carbon mobilisation from sett internode pith tissue. After 42 days growth 99.0 ± 0.72% of sett internode pith sucrose was depleted and 2.66 ± 0.16 g of new tissue accumulated. Comparison with a control treatment in which axillary buds were excised at day zero demonstrated that carbon mobilisation was driven by the accumulation of new biomass. Profiling of soluble carbohydrates (viz. sucrose, glucose and fructose), starch, total soluble protein, total amino nitrogen, free amino acids and total insoluble material showed that the sucrose stored in the sett internode pith was the only available carbon source of sufficient size at day zero for the observed biomass accumulation. Other metabolites mobilised were glucose, fructose and some amino acids, notably isoleucine and leucine that were depleted in shoot treatment setts at day 42.     

Effects of gibberellin on the cellular dynamics of dwarf pea internode development

This study analysed the dynamics of cell production and extension, and how these were affected by applied gibberellic acid (GA₃), during internode development in dwarf peas (Pisum sativum L. cv. Meteor). Image analysis was used to obtain cell number and length data for entire cell columns along the epidermis, the two outermost cortical layers, and the pith, from internode 7, over a time period covering the whole of the internode's growth phase. For a few days following the inception of an internode at the shoot apex, little further growth occurred, and there was no significant effect of GA₃ on cell division or cell extension. The subsequent growth of the internode was stimulated more than fourfold by GA₃ as a result of the production of more than twice the number of cells, which were twice as long. At least 96.5% of the cells of the mature internode were actually formed within the internode itself during this period of growth, demonstrating that the internode cells themselves represent the morphogenetic site of response to GA₃. Mitoses and cell extension occurred along the full length of the internode throughout its development. The daily changes in cell numbers were modelled by the Richards function, and manipulations of the fitted functions to reveal time trends of absolute and specific cell production rates were performed for each stem tissue. The increase in cell numbers in the +GA₃ plants was brought about by an increase in the rate of cell production, over a shorter time interval; specific cell production rates declined continuously from initial rapid rates in the +GA₃ epidermis and pith, but declined more slowly in the cortex. The control (−GA₃) epidermis and cortex cells exhibited a constant specific cell production rate (i.e. purely exponential) for several days. Cell extension rates were calculated so as to compensate for the size-reduction effects of concurrent cell division. These calculations confirmed that `real' cell extension rates were higher in the +GA₃ internodes. Models of the cellular controls of internode growth, based on the estimated dynamics of cell division and extension, are discussed. Received: 1 July 1997 / Accepted: 30 July 1997     

SHOOT GROWTH AND HISTOGENESIS OF TREES POSSESSING DIVERSE PATTERNS OF SHOOT DEVELOPMENT

Shoot growth and histogenesis were followed in five unrelated tree taxa possessing inherently diverse patterns of shoot development. Following the resumption of growth in spring, each species differs quantitatively in the number of internodes elongating contemporaneously, in rates and duration of internodal elongation and seasonal periodicity of shoot growth. The basic pattern of internode elongation and histogenesis is qualitatively similar in each of the dicotyledonous species observed irrespective of growth habit or final form of the shoot produced. During the intial phase of internode development, growth is essentially uniform throughout young internodes, corresponding to an active period of cell division during which time pith cells increase in size to about one-third their final length. Subsequently, the pattern of cell division shifts progressively upward concomitant with increased elongation and maturation of pith cells in the basal portion of developing internodes. Thereafter, a wave of cell division accompanied by cell elongation continues to proceed acropetally until growth finally ceases in the distal portion of each internode. As long as internode elongation continues, frequently at distances 15–20 cm below the shoot apex, cell divisions still occur in the distal growing portion. As successive portions of each internode mature acropetally, final length of pith cells becomes relatively uniform throughout the internode. During the process of internode growth and development, cell lengths increase only two- to threefold, whereas cell numbers increase ten- to 30-fold, indicating the dominant role of cell division and increases in cell number to final internode length. Morphological patterns of shoot expression associated with differences in internode lengths along the axis of either preformed or neoformed shoots, as well as sylleptic branches, are due to differences in cell number rather than final cell length. Significant variations in final internode lengths along the axis of episodic shoots, caused by either endogenous or exogenous factors, are also attributed to differences in cell number.     

Nuclear changes accompanying cell differentiation in stems of Pisum sativum L.

Summary Nuclear DNA, nuclear protein and nuclear size have been measured in cells of the cortex, pith and vascular tissue from three successive internodes in the stem of Pisum sativum. New techniques of computer-linked cytophotometry were used to measure these parameters simultaneously in both section and squash preparations. In cortical cells no endoreduplicated nuclei were seen in the internodes measured. In cortical cells from the oldest internode measured, a population of large nuclei with the 2C DNA amount was observed which was not present in the younger internodes. In the oldest pith nuclei measured a few 8C nuclei were present, but maturing pith was most characterized by increasing nuclear size and the population of nuclei accumulating with the 4C DNA amount. Polyploid nuclei were present in all of the vascular tissue measured, including the youngest internode. Maturing vascular tissue was also characterized by increasing nuclear size. Nuclear protein measurements demonstrated a close link between nuclear protein and nuclear size and suggest that increased nuclear size, with constant DNA content, may be due to increased nuclear protein. This raises the question of the nature and function of this nuclear protein, perhaps more characteristic of differentiating cells than dividing cells.To whom offprint requests should be sent     

PITH DEVELOPMENT IN NORMAL AND SHORT INTERNODE SEEDLINGS OF PRUNUS PERSICA VAR. LOVELL

Holmsen , Theodore W. (U. Florida, Gainesville.) Pith development in normal and short internode seedlings of Prunus persica var. Lovell. Amer. Jour. Bot. 47 (3): 173—175. Illus. 1960.–Investigation of pith cells of mature (no longer elongating) internodes of normal- and short-internode seedlings of Prunus persica var. ‘Lovell’ indicates that the short internode condition results largely from a suppression of cell division. The pith cells of normal seedlings were found to occur in an irregular fashion. The pith cells of short-internode seedlings were found to retain the linear order characteristic of the rib meristem. The relationship of these findings to the accepted long- and short-shoot conditions are discussed.     

Control of purple spot of loquat fruit (Eriobotrya japonica) by means of mineral compounds

Treatments to control purple spot of loquat fruit (Eriobotrya japonica) were tested based on the hypothesis that the disorder appears as a consequence of water unbalance between flesh and epidermal tissues caused by their different ability for sugar accumulation. Calcium nitrate, calcium chloride, Ca‐EDTA, ammonium nitrate and potassium nitrate at a concentration of 150 mm applied 2 weeks before fruit colour break reduced significantly the proportion of purple‐spotted fruit, giving rise to a reduction of water potential of the epidermal tissue that allows it to retain water. The most favourable date of treatment was during the 2 weeks prior to fruit colour break.     

POLAR TRANSPORT OF GROWTH-REGULATORS IN PITH AND VASCULAR TISSUES OF COLEUS STEMS

Movement of IAA-C¹⁴ and 2,4-D-C¹⁴ through cylinders of known size and histology was compared using liquid scintillation counting. Both auxins showed strongly polar movement, even through pith parenchyma cut from Coleus internode #5, the youngest internode to have ceased elongation. The polar movement was correlated with sizable elongation of the excised cylinders. Velocities of basipetal movement for a given auxin, as determined by the intercept method, showed small or negligible differences between pith and “corner” cylinders. (Corner cylinders comprised mostly vascular tissue, plus some cortical, pith, and epidermal cells.) For IAA, basipetal velocities ranged from 2.1 to 3.3 mm per hr; for 2,4-D, they were 0.6–0.8. For both auxins there was much more net loss into corner than into pith cylinders, a difference associated with the fact that corner cylinders showed 10 times as many cells in transection. More 2,4-D moved basipetally through corner than through pith cylinders and the reverse was true of IAA. By chromatographic evidence, all the radioactivity in the basal receiving blocks was still associated with the auxin molecules.     

Ultrastructure of fibre and parenchyma cell walls during early stages of culm development in Dendrocalamus asper

BACKGROUND AND AIMS: The anatomy of bamboo culms and the multilayered structure of fibre cell walls are known to be the main determinant factors for its physical and mechanical properties. Studies on the bamboo cell wall have focussed mainly on fully elongated and mature fibres. The main aim of this study was to describe the ultrastructure of primary and secondary cell walls in culm tissues of Dendrocalamus asper at different stages of development. METHODS: The development of fibre and parenchyma tissues was classified into four stages based on light microscopy observations made in tissues from juvenile plants. The stages were used as a basis for transmission electron microscopy study on the ultrastructure of the cell wall during the process of primary and early secondary cell wall formation. Macerations and phloroglucinol-HCl staining were employed to investigate fibre cell elongation and fibre cell wall lignification, respectively. KEY RESULTS: The observations indicated that the primary wall is formed by the deposition of two distinct layers during the elongation of the internode and that secondary wall synthesis may begin before the complete cessation of internode and fibre elongation. Elongation was followed by a maturation phase characterized by the deposition of multiple secondary wall layers, which varied in number according to the cell type, location in the culm tissue and stage of shoot development. Lignification of fibre cell walls started at the period prior to the cessation of internode elongation. CONCLUSIONS: The structure of the primary cell wall was comprised of two layers. The fibre secondary cell wall began to be laid down while the cells were still undergoing some elongation, suggesting that it may act to cause the slow-down and eventual cessation of cell elongation.     

Vertical differences in Phragmites australis culm anatomy along a water depth gradient

We reported on quantitative anatomical comparisons of reed culms grown under a wide range of water depth (from −10 to +230 cm). The study focused mainly on the above water parts of ramets, but also provided an insight into the submerged internodes. Investigated anatomical features were: radial thickness of the internode wall and that of each tissue constituting it (epidermis and subepidermal tissues, aerenchyma channels, cortical sclerenchyma and parenchyma); areas of aerenchyma channels and the summed transversal area of them; areas of the innermost vascular bundles involving the bundle sheath sclerenchyma, phloem and metaxylem vessels; the cross-sectional area of the pith cavity and that of the internode wall. After a 2–3% decrease below the uppermost internode, the thickness of internode tissues, except for cortical sclerenchyma, continuously increased downwards. Quantitative differences between the ramets grown at different water depths were observed only from the lower aerial internodes. Parenchyma taking part considerably in the mechanical stability of culms with its large extension and thick cell walls was more-layered and thicker (with the maximum thicknesses of approximately 1200 and 1000 μm) in ramets from 180–200 than from 20–30 cm water depth. Areas of the innermost vascular bundles increased downwards and decreased after a maximum value appearing in lower internodes of ramets grown in deeper than shallower water. Aerenchyma channels appeared farther up from the water surface in culms in shallower than deeper water: the top ends were found about 50 cm higher in culms from 20–30 than from 180–200 cm water depth. Along the transect at right angles to the shore, the radial thickness and area of aerenchyma channels were higher in ramets grown in medium water depth—the summed transversal area of them was greater than 4 mm² in internodes at the water surface, while plants from the two ends of the water depth gradient had smaller aerenchyma channels (with approximately 1 mm² total areas). In contrast, pith cavity area at the water surface continuously increased – up to 60 mm² – from shallower to deeper water. Therefore, oxygen transport in ramets grown at the open water fringe of stands may occur mostly in the pith cavity.     

Internode morphometrics and allometry of Tonkin Cane Pseudosasa amabilis

Pseudosasa amabilis (McClure) (Poales: Gramineae) is a typical bamboo species naturally distributed in large area of south China and famous for its culm strength. Although bamboos were found to share the same development rule, the detailed internode morphology of bamboo culm was actually not fully expressed. We explored internode morphology of P. amabilis using 11 different physical parameters in different dimensions (1–4). As Taylor's power law (TPL) is generally applicable to describe relationship between mean and variance of population density, here we used TPL to evaluate the differences between internodes, and further, the relationship between dimension and TPL. Results showed that length (L), hollow radius (HR), hollow area (HA), hollow cylinder volume (HCV), total cylinder volume (TCV), density (De), and weight (W) all presented positive skewed distribution in varying degrees. For the basic one‐dimensional parameters, the 9th internode was the longest, the 7th the heaviest, while thickness (T) decreased with internodes. Diameter (D) decreased in general but with an inconspicuous local mode at the 5–6th internodes, potentially due to the rapid height growth. The longest (9th) internode was the “turning point” for T‐D and HR‐D relationships. Scatter plot changing trends of W to the one‐dimensional parameters after the heaviest (7th) internode were reversed, indicating a deceleration of growth speed. TPL was not holding well in one‐dimensional parameters (R²: 0.5413–0.8125), but keep increasing as the parameter's dimension increasing (R² > 0.92 for two‐dimensional, R² > 0.97 for three‐dimensional, and R² > 0.99 for four‐dimensional parameters.), suggesting an emergence mechanism of TPL related to both the physical dimensions of morphological measures and the allometric growth of bamboo. From the physical fundamental level, all existences are the expression of energy distribution in different dimensions, implying a more general rule that energy distribution holds better TPL in higher dimension level.     

CAVITY FORMATION IN WINTER BUDS OF PINUS BANKSIANA

The formation of a cavity between the rib meristem and the pith in winter buds of Pinus banksiana is described. Necrosis of cells at the juncture of the rib meristem and pith begins in November, and by mid-March the necrotic region enlarges, forming a cavity that separates the rib meristem from the pith. The cavity appears to be formed by autolysis, rather than a pulling apart of the cells. The function of the cavity remains speculative.     

The influence of intermittent soil drying on growth, cell number, and final cell length in the pith of mature internodes in Helianthus annuus L. and Liquidambar styraciflua L.

The sensitivity of cell division in developing internodes to plant water deficits has not been previously documented. In this study two diverse taxa, Helianthus annuus L. and Liquidambar styraciflua L. were chosen because cell divisions in the pith and cortex continue to occur acropetally throughout the period of internode elongation. Potted plants were given 6-d cycles of soil drying between waterings to observe the effects of moderate, intermittent water deficits on final cell pattern in developing internodes. Under this regime, internode and leaf growth were inhibited although leaf and shoot turgidity were restored daily by nocturnal rehydration. The percent inhibition of final internode lengths was similar in both taxa, increasing from 23–58% in contemporaneously developing internodes. Of this inhibition, 9–48% in H. annuus compared with 97–100% in L. styraciflua was attributable to decreases in cell number in mature internodes. While cell divisions were severely inhibited in both taxa, differences in sensitivity appear related to differences in patterns of histogenesis associated with pronounced inherent differences in final cell lengths. Final cell lengths in H. annuus exceed those of L. styraciflua by 7–8-fold and can play a more dominant role in final internode lengths than total cell number. Conversely, in L. styraciflua total cell number, rather than final cell length, accounts for most of the variation in final internode length. These studies demonstrate species differences in sensitivity of cell division in developing internodes to intermittent water deficits.     

Mode of Infection of Ascochyta Blight of Chickpea Caused by Ascochyta rabiei

Process of infection and histological changes with Ascochyta blight of chickpea caused by A. rabiei (Pass.) Labr. were studied by light microscopy. Germ tubes from conidia of the fungus penetrate the stem tissue at the juncture of two epidermal cells and form subepidermal aggregates until the fourth day. On the sixth day, yellowing and necrotisation of host tissue coincides with formation of mature pycnidia. Fungus causes extensive damage to cellulosic cell walls of parenchymatous cortical and pith tissues in advance of invading hyphae indicating involvement of cell wall degrading enzymes. Lignified tissues, particularly xylem tracheary elements, remain intact.