Cell Division Phase Research Articles

Molecular cloning of the Notophthalmus viridescens radical fringe cDNA and characterization of its expression during forelimb development and adult forelimb regeneration.

Larval and adult newts provide important experimental models to study limb development and regeneration. These animals have exceptional ability to regenerate their appendages, as well as other vital structures. Our research examines the role of the fringe gene (fng) in the developing and regenerating adult newt forelimb. Fringe codes for a secretory protein. It was first discovered in Drosophila, and later homologues were isolated in Xenopus laevis, chick and mouse. This gene has been highly conserved throughout evolution, indicating its crucial role in vertebrate and invertebrate development. We have isolated, cloned, and sequenced the full length of the Notophthalmus viridescens radical fringe cDNA (nrFng) by screening a newt forelimb blastema cDNA library with a 500-bp fragment of the Xenopus lunatic fringe cDNA. The newt fringe cDNA codes for a 396 amino acid protein with a predicted N-terminal signal sequence. Newt fringe shows high homology with radical fringe homologues of many species. Whole mount mRNA in situ hybridization on several stages of newt limb development reveals that nrFng is first expressed in the limb field, with intense expression as the limb bud develops. However, gene expression diminishes with more advanced digit development. A significant role in adult forelimb regeneration is also evident, as we isolated the cDNA from a regeneration-specific library and found it highly expressed during the regenerative phases of active cell division and then down regulated at sites undergoing differentiation and morphogenesis.

Phytotoxic lignans of Leucophyllum frutescens.

Bioassay-guided fractionation of the hexane:ethyl acetate (1:1) extract of the leaves of Leucophyllum frutescens (Berl.) I.M.Johnst (Scrophulariaceae) led to the isolation of its phytotoxic constituents diayangambin (1), epiyangambin (2), diasesartemin (3) and epiashantin (4). Phytotoxicity was demonstrated as inhibition of seed germination of Agrostis stolonifera cv. penncross (Poaceae) and inhibition of development of Lactuca sativa L. (Asteraceae) seedlings in a microassay using 24-well plates. Compound 1 was the most phytotoxic to L. sativa, showing strong inhibitory activity at 110 microM. Compound 1 was more active than 2 and 3 in inhibiting the growth of A. stolonifera with I(50) values of 160, 670 and 930 microM, respectively. At a concentration of 500 microM, these compounds inhibited all phases of onion root cell division. This is the first demonstration of antimitotic activity of these furofuran lignans, and the first report of their isolation from this species.

Cytometric measurement of the DNA cell cycle in the presence of chlorophyll autofluorescence in marine eukaryotic phytoplankton by the blue-light excited dye YOYO-1

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 185:301-307 (1999) - doi:10.3354/meps185301 Cytometric measurement of the DNA cell cycle in the presence of chlorophyll autofluorescence in marine eukaryotic phytoplankton by the blue-light excited dye YOYO-1 Frank J. Jochem1,*, Doris Meyerdierks2 1Institut für Meereskunde, Düsternbrooker Weg 20, D-24105 Kiel, Germany 2Universität Bremen, FB II Meeresbotanik, Postfach 330440, D-28334 Bremen, Germany *Present address: The University of Texas at Austin, Marine Science Institute, 750 Channel View Drive, Port Aransas, Texas 78373, USA. E-mail: frank@emcs.de ABSTRACT: The novel blue-light excited fluorescent DNA dye YOYO-1® was tested on cultures of 8 eukaryotic phytoplankton species from the classes of Prymnesiophyceae, Bacillariophyceae, Coscinodiscophyceae, Prasinophyceae and Dinophyceae, and the coccoid cyanobacterium Synechococcus sp. In all tested species, YOYO staining of formalin fixed cells allowed the differentiaton of cell cycle phases of G1, S, and G2+M in the presence of chlorophyll autofluorescence in flow cytometric analyses. Diel cell cycle patterns of Emiliania huxleyi and Skeletonema costatum grown under a light:dark cycle and under continuous light could be established. Under the light/dark cycle, DNA replication occurred during the night and cell division in the early morning in both species. However, S. costatum spent a longer fraction of their cell cycle in S and G2+M phases than E. huxleyi did. Changes in cellular chlorophyll content and cell size could be observed in relation to cell cycle phasing. Cell division phasing was nearly lost under continuous light. Growth rates calculated from DNA cell cycle analyses agreed well with those established from cell counts. KEY WORDS: Cell cycle · Cytometry · DNA · Growth rates Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 185. Publication date: August 20, 1999 Print ISSN:0171-8630; Online ISSN:1616-1599 Copyright © 1999 Inter-Research.

Early- and mid-season temperature effects on the growth and composition of satsuma mandarins

SummaryThe growth and composition of satsuma mandarin fruit from trees which had been covered by a tunnel house during either stage one (the first ten weeks after anthesis), stage two (10±20 weeks after anthesis) or throughout fruit growth, were compared with those grown under ambient conditions. During each stage temperature was identified as the key factor influencing fruit development and changes to fruit were generally maintained when trees were returned to ambient conditions. Raising maximum air temperatures in the canopy of trees by 2.4.K during stage one increased both fruit growth rates and accumulation of sugars throughout their development. Titratable acidity levels were also increased and quinate levels decreased when trees were warmed during stage one, but these differences did not persist until harvest. Fruit grown under elevated temperatures during stage one had larger pedicels and dorsal vascular bundles, which suggests that temperatures during early fruit development determine the fruits' subsequent ability to import and unload carbohydrate. Raising canopy air temperatures during the second stage of fruit growth lowered tritratable acidity, total acids, citrate and malate levels compared with those in control fruit, and these lower levels were maintained until harvest. Fruit from trees grown in a tunnel house all season and those from control trees were supplied with photosynthate synthesized from 14CO2 or injected with 14C sucrose during either stage one or stage two of fruit development. During stage one, warmed fruit incorporated less radioactivity into structural components and quinate, and more into other soluble components in juice sacs. This suggests that elevated temperatures advanced early fruit development and shortened the duration of the cell division phase. During stage two, less radioactivity was incorporated into acids and more into sugars in warmed fruit.

Modelling and analysis of time-lags in some basic patterns of cell proliferation.

In this paper, we present a systematic approach for obtaining qualitatively and quantitatively correct mathematical models of some biological phenomena with time-lags. Features of our approach are the development of a hierarchy of related models and the estimation of parameter values, along with their non-linear biases and standard deviations, for sets of experimental data. We demonstrate our method of solving parameter estimation problems for neutral delay differential equations by analyzing some models of cell growth that incorporate a time-lag in the cell division phase. We show that these models are more consistent with certain reported data than the classic exponential growth model. Although the exponential growth model provides estimates of some of the growth characteristics, such as the population-doubling time, the time-lag growth models can additionally provide estimates of: (i) the fraction of cells that are dividing, (ii) the rate of commitment of cells to cell division, (iii) the initial distribution of cells in the cell cycle, and (iv) the degree of synchronization of cells in the (initial) cell population.

Transient Increase in Cerebellar Transcriptional Activity Precedes the Expression of GABAA Receptor α6 Subunit mRNA during Postnatal Maturation

The purpose of this study was to investigate the postnatal expression of GABA_A receptor α6 subunit genes in the context of cerebellar differentiation. We examined steady-state levels of GABA_A receptor α1 and α6 subunit mRNAs, polyadenylated (polyA⁺) mRNA and β-actin mRNA in7-, 14-, 21-, 28-, 35-, 49- and 120-day-old rats. Messenger RNA expression and splicing were evaluated in parallel using Northern blot analysis and in situ hybridization histochemistry. The expression of mature GABA_A receptor α6 subunit mRNA species (2.7 kb) was found 1 week after birth in cerebellar granule cells. Prior to stable expression of the mature α6 subunit gene, we detected large α6 subunit premessengers (3.8 and 3.5 kb) by Northern blot analysis. These premessenger species were detected in prenatal day (PND) 15 and neonatal rat cerebellum, when the mature α6 subunit mRNAs (2.7 kb) were not yet expressed. The maximal expression of mature α6 subunit mRNA species was observed at PND 21 when the peak level of cerebellar transcriptional activity was measured by polyA⁺ RNA levels. In contrast, β-actin mRNA expression was decreased at PND 21 compared to birth levels. These major transcriptional events take place during a period of about 1 week (between PND 14 and 21), immediately following the most active phase of cell division in the external granule layer and migration of granule cells to the internal granule cell layer. Comparison between the relative abundance of these genes shows that differential regulation of each gene occurs during postnatal development. The induction of GABA_A receptor α6 subunit gene expression is preceded by a reduction in β-actin mRNA levels and a transient increase in total transcriptional activity. The expression of α6 subunit mRNA is maintained at the PND 21 level through adulthood, but the α1 subunit mRNA levels decrease drastically within the following week (from PND 21 to 28). These results suggest that tissue-specific expression of the GABA_A receptor α6 subunit gene is correlated with a series of developmentally regulated morphologic and transcriptional events.

Impact of Temperature on Apple Fruit Growth

Apple fruitlet growth responses to temperature were studied, for different durations following bloom (DAFB), under controlled environment (CE) conditions. Container-grown trees of `Red Delicious', `Golden Delicious', `Braeburn', `Fuji', and `Royal Gala' were placed in different maximum/minimum temperature regimes, ranging from 9/3 to 25/15°C for various periods, including 10–40, 10–80, and 40–80 DAFB. Temperature treatments were selected to identify possible differences between mean and maximum/minimum differential effects Trees were placed outdoors following the CE treatment to allow impacts on subsequent fruit development to be determined. The impact of temperature was dramatic. For example, fruit expansion rate for `Red Delicious' varied from 0.12 mm/day at 9/3°C to 0.98 mm/day at 25/15°C. Furthermore, the cell division phase was considerably longer under cooler temperatures. The influence of post-bloom temperature, for even short durations, was evident at harvest in both fruit size and in different fruit maturity indices. Differences in temperature sensitivity were evident amongscultivars. A detailed model has been developed to integrate the responses that have been determined.

Growth dynamics in a natural biofilm and its impact on oral disease management.

Measurements of the microbial growth dynamics in natural biofilm communities are almost non-existent. In a recent study, the biofilm formation on teeth was examined. A previously unknown active period of bacterial division occurred at a certain density of plaque bacteria on tooth enamel. The density-dependent cell-division phase of plaque formation contributed 90% of the biomass in the first 24 hrs of plaque formation. This suggested that growth was induced by the bacteria. In vitro assays were developed for rapid evaluation of the growth of surface-linked bacteria by the measurement of cellular components associated with growth on a per cell per time basis. Cell-free supernatants (termed START) of media in contact with bacteria were assayed for their effects on DNA synthesis and other cellular components associated with growth. START was found to increase the incorporation of [3H-methyl]-thymidine on a per cell per time basis, when compared with media not in contact with bacteria. Additional in vivo studies and in situ-based models of complex biofilms are needed if all of the mechanisms involved in the rapid accumulation of biofilm bacteria on teeth and other surfaces are to be understood.

Mathematical Modelling of the Interleukin‐2 T‐Cell System: A Comparative Study of Approaches Based on Ordinary and Delay Differential Equation

Cell proloferation and differentiation phenomena are key issues in immunology, tumour growth and cell biology. We study the kinetics of cell growth in the immune system using mathematical models formulated in terms of ordinary and delay differential equations. We study how the suitability of the mathematical models depends on the nature of the cell growth data and the types of differential equations by minimizing an objective function to give a best‐fit parameterized solution. We show that mathematical models that incorporate a time‐lag in the cell division phase are more consistent with certain reported data. They also allow various cell proliferation characteristics to be estimated directly, such as the average cell‐boubling time and the rate of commitment of cells to cell division. Specifically, we study the interleukin‐2‐dependent cell division of phytohemagglutinin stimulated T‐cells — the model of whic can be considered to be a general model of cell growth. We also review the numerical techniques available for solving delau differential equations and calculating the least‐squares best‐fit parameterized solution.

Sequestration of type VI collagen in the pericellular microenvironment of adult chondrocytes cultured in agarose

The chondron represents the chondrocyte and its pericellular microenvironment and plays an important role in the progression of osteoarthritis. Type VI collagen is preferentially localized in the pericellular microenvironment of adult articular cartilage and increases during osteoarthritis. In this study, we characterized the pericellular sequestration of type VI collagen in long-term chondrocyte-agarose cultures, and assessed the action of interleukin-1 on type VI collagen deposition and assembly. Immunohistochemical and biochemical analysis showed that cultured chondrocytes initiate type VI collagen sequestration immediately upon plating and continue pericellular matrix sequestration in a time dependent manner. Confocal microscopy confirmed the cell surface localization and pericellular accumulation of type VI collagen, while image analysis identified a 'cargo-net like' organization of type VI collagen around each chondrocyte. Quantitative analysis revealed a primary phase of rapid cell division and low levels of type VI collagen sequestration, followed by a secondary phase of relative growth stability and high levels of type VI collagen deposition. Interleukin-1 treated cultures showed increased sequestration and retention of type VI collagen in an expanded microenvironment surrounding the chondrocytes. The data suggests a role for type VI collagen in the differentiation of the pericellular microenvironment in vitro. The increased type VI collagen sequestration promoted by interleukin-1 was consistent with previous studies on osteoarthritic cartilage, and implies a functional role for type VI collagen in the chondron remodeling associated with cartilage degradation.

Two Critical Periods of Sonic Hedgehog Signaling Required for the Specification of Motor Neuron Identity

Antibodies that block Sonic Hedgehog (SHH) signaling have been used to show that SHH activity is required for the induction of floor plate differentiation by the notochord and independently for the induction of motor neurons by both the notochord and midline neural cells. Motor neuron generation depends on two critical periods of SHH signaling: an early period during which naive neural plate cells are converted into ventralized progenitors and a late period that extends well into S phase of the final progenitor cell division, during which SHH drives the differentiation of ventralized progenitors into motor neurons. The ambient SHH concentration during the late period determines whether ventralized progenitors differentiate into motor neurons or interneurons, thus defining the pattern of neuronal cell types generated in the neural tube.

Controlling seed development and seed size in Vicia faba: a role for seed coat‐associated invertases and carbohydrate state

We previously provided evidence that seed coat‐associated invertase is involved in controlling the carbohydrate state of developing seeds and, by this way, triggering developmental processes (Weber et al. (1995) Plant Cell, 7, 1835–1846). To verify our postulate, we compared seed development of two genotypes of Vicia faba differing in seed weight. The seed coat of the large‐seeded genotype formed a higher number of parenchymatous cell layers and matured later. VfCWINV1 encoding a cell wall‐bound invertase is expressed in the unloading zone of the seed coat. mRNA levels peaked later in ‘large’ coats and mRNA was present in more cell layers over a longer time period. Cell wall‐bound invertase activity revealed a similar accumulation pattern, obviously generating the high hexose conditions present in the endospermal cavity bathing the premature cotyledons and thus controlling their carbohydrate state. High hexose conditions were correlated with an extended mitotic activity of the ‘large’ cotyledons. In ‘large’ and ‘small’ cotyledons, sucrose levels rose when hexoses decreased apparently terminating cell divisions and initiating differentiation and storage activities. This developmental switch was delayed in ‘large’ embryos. To prove the outlined relationship, sucrose was added in vitro to mitotically active cotyledons. This treatment favoured nuclear expansion and starch accumulation over cell division. In contrast, a hexose‐based medium maintained cell divisions. We conclude that development of the embryo is coordinately regulated with that of the maternal seed coat which controls, by metabolic signals, the phase of cell division of the embryo and consequently also seed size.

Silver Staining of Nucleolar Organizer Regions

The use of silver staining to identify nucleolar organizer regions (NORs) has increased dramatically in recent years. NORs are cellular structures present during the mitotic and G1 phases of cell division. The number and size of silverstained NORs (AgNORs) have been associated with increased rates of proliferation and decreased nuclear stability. The clinical utility of the evaluation of AgNORs in the differential diagnosis and prognosis of cancer is currently being studied. Most histology laboratories are equipped to perform the staining procedure, which is both simple and inexpensive. Routine formalin fixation and staining times that do not exceed 30 min are satisfactory for most tissues. Although computer-based image cytometry generates objective analyses that differentiate subtle differences in the expression of AgNORs, the types of patterns of interest, the variation of expression between tissues, and the number of AgNORs to measure per case have not yet been established. This lack of standardization has led to contradictory data concerning the clinical utility of the evaluation of AgNORs. In non-Hodgkin's lymphoma, bladder cancer, malignant melanoma, and brain cancer, however, the number of AgNORs more often correlates with the prognosis of the patient than with staging of the lesions. In other cancers, including prostate cancer, a consistent correlation of the number of AgNORs with prognosis has not been shown. Future investigations using standardized measurement protocols and analyses of the size, number, and shape of AgNORs compared to molecular activity events in the nucleus may offer more significant test outcomes. From those findings, new measurement algorithms may be developed to more accurately measure and assess the usefulness of AgNORs in situ. (The J Histotechnol 19:241, 1996)

Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). I. Changes during fruit development

The cytokinin content in fruit tissue of the kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) was monitored during fruit development to identify which cytokinins were present and if they were linked with specific stages of fruit growth. Cytokinins were isolated and purified by column chromatography and high‐performance liquid chromatography and quantified by radioimmunoassay. A novel HPLC step utilising an amine column was successfully introduced as a preparative step in the separation of the O‐ and 9‐glucosides from the free bases and ribosides. The radioimmunoassay results were validated, and the different cytokinins identified, by gas chromatography‐mass spectrometry. Cytokinins detected in fruit included the cytokinin free bases, zeatin and isopentenyladenine, their ribosides, nucleotides and both O‐ and 9‐glucosides. Both qualitative and quantitative changes of the cytokinins occurred during fruit development. A decrease in cytokinin concentration occurred after anthesis (from 342 pmol g−1 fresh weight at anthesis to 41 pmol g−1 fresh weight 27 days after anthesis). A large increase in cytokinin concentration and content per fruit occurred as the fruit reached commercial maturity (to 1900 pmol g−1 fresh weight). Individual cytokinins showed quite different patterns. Zeatin, in particular, showed a peak in concentration (13 pmol g−1 fresh weight) 11 days after anthesis that correlated with the beginning of the cell division phase of fruit growth. The accumulation of cytokinin (mostly zeatin riboside or zeatin nucleotide) in mature fruit may be of significance for the postharvest storage of kiwifruit fruit.

A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division and secretion in surface membrane vesicles

A 26-kDa murein hydrolase is the major autolysin of Pseudomonas aeruginosa PAO1, and its expression can be correlated with the growth and division of cells in both batch and synchronously growing cultures. In batch cultures, it is detected primarily during the mid-exponential growth phase, and in synchronous cultures, it is detected primarily during the cell elongation and division phases. Immunogold labeling of thin sections of P. aeruginosa using antibodies raised against the 26-kDa autolysin revealed that it is associated mainly with the cell envelope and in particular within the periplasm. It is also tightly bound to the peptidoglycan layer, since murein sacculi, isolated by boiling 4% sodium dodecyl sulfate treatment, could also be immunogold labeled. Since division is due to cell constriction in this P. aeruginosa strain (septa are rarely seen), we cannot comment on the autolysin's contribution to septation, although constriction sites were always heavily labeled. Some labeling was also found in the cytoplasm, and this was thought to be due to the de novo synthesis of the enzyme before translocation to the periplasm. Interestingly, the autolysin was also found to be associated with natural membrane vesicles which blebbed from the surface during cell growth; the enzyme is therefore part of the complex makeup of these membrane packages of secreted materials (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). The expression of these membrane vesicles was correlated with the expression of B-band lipopolysaccharide.

Model of Leaf Area Expansion in Field Pea Subjected to Soil Water Deficits

AbstractYield of field pea (Pisum sativum) is sensitive to soil water deficits. An important component of this sensitivity is the response of leaf area to drought. A simple, mechanistic model was developed to allow predictions of leaf area development in response to soil water deficits. The model calculates leaf areas of individual drought‐stressed leaves relative to leaves on well‐watered plants as the product of the number of cells and the size of the cells. Therefore, distinct phases of cell division and cell expansion were identified and temporally distinguished in the model. Cell division and cell expansion in the model were both sensitive to the fraction of transpirable soil water. The model proved capable of predicting the temporal development and the final area (r2 = 0.91) of individual leaves on pea plants in response to soil water deficits.

Mitotic mechanisms in Alzheimer's disease?

The mechanism(s) leading to widespread hyper-phosphorylation of proteins in Alzheimer's disease (AD) are unknown. We have characterized seven new monoclonal antibodies recognizing independent phospho-epitopes in the paired helical filament proteins (PHF) found in AD brain. These antibodies show pronounced immunoreactivity with cultured human neuroblastoma cells that are in the M phase of cell division, but have no discernible reactivity with interphase cells. Immunoreactivity with these antibodies does not localize to the microtubule spindles or chromosomes in M phase, but is confined to the surrounding cytoplasm. Similar staining in M phase is observed with cultured cells of various tissue types and species. Cells arrested in M phase with the microtubule depolymerizing agent, nocodazole, show marked increases in immunoreactivity with the antibodies by immunofluorescence staining, ELISA, and immunoblotting. In neuroblastoma cells, the appearance of the TG/MC phospho-epitopes coincides with activation of mitotic protein kinases, but not with the activity of the neuronal specific cyclin-dependent kinase, cdk5. These data suggest that the TG/MC epitopes are conserved mitotic phospho-epitopes produced as a result of increased mitotic kinase activity. To investigate this possibility in AD, we examined the staining of human brain tissue with MPM-2, a marker antibody for mitotic phospho-epitopes. It was found that MPM-2 reacts strongly with neurofibrillary tangles, neuritic processes, and neurons in AD but has no staining in normal human brain. Our data suggest that accumulation of phospho-epitopes in AD may result from activation of mitotic posttranslational mechanisms which do not normally operate in mature neurons of brain.

Immunohistochemical detection of CDK4 and 9 16INK4proteins in cutaneous malignant melanoma

p16INK4 gene, which encodes a specific inhibitor of cyclin-dependent kinase 4 (CDK4), has been recently reported as an important tumour suppressor gene. It is mapped to chromosome 9p21, which is frequently deleted or mutated in many tumour cell lines including malignant melanoma. Since the CDK4/cyclin D complex propels a cell to go through the G1 check point of the cell cycle, a critical phase of cell division, alteration of the p16INK4 gene could lead a cell to uncontrolled proliferation and malignant transformation. To clarify any role for p16INK4 and CDK4 proteins in the development of human malignant melanoma, we have examined, immunohistochemically, the expression of these two proteins in melanocytic neoplasms including 19 primary lesions of non-familial melanoma. Intense nuclear and/or cytoplasmic expression of the CDK4 protein was observed in 11 of 19 cases (58%) of melanoma. In contrast, virtually no nuclear or cytoplasmic staining for CDK4 protein was detected in 28 benign melanocytic naevi, including six Spitz naevi. Expression of p16INK4 protein was observed in three of 19 melanomas (16%) and in 17 of 28 benign naevi (61%). Inverse expression of CDK4 and p16INK4, at individual cell level, was detected in one case of melanoma. The present study suggests that CDK4 overexpression is characteristic for malignant melanoma, and probably reflects its autonomous accelerated cell proliferation. The expression rate of p16INK4 protein in malignant melanoma was lower than that in benign naevi, although the significance of p16INK4 deletion in melanoma development has not been definitely confirmed.

Mechanism for increased leaf growth in elevated CO2

The effect of exposure to elevated CO2 on the processes of leaf cell production and leaf cell expansion was studied using primary leaves of Phaseolus vulgaris L. Cell division and expansion were separated temporally by exposing seedlings to dim red light for 10 d (when leaf cell division was completed) followed by exposure to bright white light for 14 d (when leaf growth was entirely dependent on cell expansion). When plants were exposed to elevated CO2 during the phase of cell expansion, epidermal cell size and leaf area development were stimulated. Three pieces of evidence suggest that this occurred as a result of increased cell wall loosening and extensibility, (i) cell wall extensibility (WEx, measured as tensiometric extension using an Instron) was significantly increased, (ii) cell wall yield turgor (V, MPa) was reduced and (iii) xyloglucan endotransglycosylase (XET) enzyme activity was significantly increased. When plants were exposed to elevated CO2 during the phase of cell division, the number of epidermal cells was increased whilst final cell size was significantly reduced and this was associated with reduced final leaf area, WEx and XET activity. When plants were exposed to elevated CO2 during both phases of cell division and expansion, leaf area development was not affected. For this treatment, however, the number of epidermal cells was increased, but cell expansion was inhibited, despite exposure to elevated CO2 during the expansion phase. Assessments were also made of the spatial patterns of WEx across the expanding leaf lamina and the data suggest that exposure to elevated CO2 during the phase of leaf expansion may lead to enhanced extensibility particularly at basal leaf margins which may result in altered leaf shape. The data show that both cell production and expansion were stimulated by elevated CO2, but that leaf growth was only enhanced by exposure to elevated CO2 in the cell expansion phase of leaf development. Increased leaf cell expansion is, therefore, an important mechanism for enhanced leaf growth in elevated CO2, whilst the importance of increased leaf cell production in elevated CO2 remains to be elucidated.

Defoliation increases fruit abscission and reduces carbohydrate levels in developing fruits and woody tissues of Citrus unshiu

Carbohydrate levels in fruitlets of control and defoliated trees of dwarf Citrus unshiu (cvs. Clausellina and Okitsu) were determined from bloom up to the end of fruit set. Full and partial (50%) defoliations were carried out at anthesis and at the beginning of the cell enlargement period. Carbohydrate reserves in woody storage tissues were also analyzed soon after defoliation. In control fruits, sucrose, glucose, fructose and starch showed a transient increase at anthesis and remained low during the cell division phase. Soluble sugars accumulated at the onset of the cell enlargement phase. Defoliation did not modify carbohydrate status in either fruits or woody tissues during the cell division period, although the first fruit abscission wave, which takes place at this stage, was increased. Fruit growth was not altered either. However, at the onset of the second phase of growth, soluble sugars were reduced in fruits and sucrose and starch were lower in woody tissues from defoliated plants. In these plants, fruit abscission at this stage (June drop) was also increased, while fruit growth was arrested or delayed. Once the June drop was overcome, fruits remaining on either defoliated or control plants were similar in size and contained analogous high levels of sugars. Taken together, the results suggest that developing fruitlets are utilization sinks during the cell division period and act as storage sinks during the cell enlargement stage. At this critical transition, sucrose levels correlated positively with fruit growth and negatively with fruit abscission. These results are compatible with the proposal that sucrose supply is a major factor of the regulatory mechanism for citrus fruit abscission during the June drop.