The cellular parameters of leaf development in tobacco: a clonal analysis

Cell division and cell death in the cell division zone of the roots of rye seedlings under salt stress were analyzed quantitatively. Cell division was examined by immunological staining with anti-5-bromo-2'-deoxyuridine (BrdU) and cell death by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). In the 0-700 µm portion from the root tip, which is the cell division zone, the frequency of cell division increased linearly during BrdU treatment. Therefore, the frequency of cell division under salt stress was compared with that in the control at 7 hours after application of BrdU. In the 250 mM NaCl solution (salt stress), the frequency of cell division was decreased and that of cell death was increased as compared with the control resulting in the inhibition of root elongation. In the presence of 50 and 100 mM NaCl, the frequency of cell division was also significantly increased and cell death was hardly detected, and root growth was unchanged as compared with the control. These results suggested that the increase of cell division complemented the decrease of cell elongation due to salt stress, and consequently maintained root growth under mild salt stress conditions.

In young plants of Salix pentandra, a temperate zone deciduous woody species, elongation growth ceases and a terminal bud is formed at day lengths shorter than a critical length. This is the first step in dormancy development, making survival under harsh winter conditions possible. Early studies strongly indicate that gibberellin is involved in the photoperiodic control of bud set and bud break. GA(1) action was studied by application under short days to plants where cessation of shoot elongation had occurred, followed by subsequent anatomic investigations of shoot tips. Under short days the frequency of cell division decreased rapidly along with the earlier observed decrease in GA(1) levels. Application of GA(1) to short-day-induced terminal buds rapidly stimulated cell division in apices several days before visible shoot elongation in response to this treatment was observed. One day after GA(1) application a fourfold increase in cell division frequency in apices was observed, increasing to a maximum of sevenfold 2 days after application. Long-day treatment leading to induction of bud break after about 4-6 days was followed by slowly increasing frequency of cell divisions. In earlier studies of this species, short days and gibberellins had no effect on cell elongation. These data show that increased GA(1) content, by application or long-day treatment, results in increased frequency of mitosis. This strongly indicates that GA(1) affects stem elongation in connection with bud set and bud break primarily by affecting cell divisions in subapical tissues.

The cell population kinetics of excised, cultured pea roots was studied with the use of tritiated thymidine and colchicine to determine (1) the influence of excision, (2) the influence of sucrose concentration, (3) the average mitotic cycle duration, and (4) the duration of mitosis and the G1, S, and G2 periods of interphase.1 The results indicate that the process of excision causes a drop in the frequency of mitotic figures when performed either at the beginning of the culture period or after 100 hours in culture. This initial decrease in frequency of cell division is independent of sucrose concentration, but the subsequent rise in frequency of division, after 12 hours in culture, is dependent upon sucrose concentration. Two per cent sucrose maintains the shortest mitotic cycle duration. The use of colchicine indicated an average cycle duration of 20 hours, whereas the use of tritiated thymidine produced an average cycle duration of 17 hours.

The meristematic activity of the apical cell and its derivatives (merophytes) in the unbranched, determinate roots of Azolla filiculoides Lam. was investigated. The plane of division of the apical cell indicates that it is the initial of each merophyte. The division plane of each newly formed merophyte is strictly periclinal to the root surface and provides confirmation that the immediate derivatives of the apical cell cannot be the ultimate root initials. The frequency of cell division as determined by the mitotic index, and by the duration of the cell cycle as determined by the colchicine method, confirmed the meristematic activity of the apical cell. As roots increase in length, the duration of the cell cycle in the total meristem increases, with the apical cell possessing the longest cell cycle, whereas the immediate derivatives maintain approximately the same cycle duration as in shorter roots. In determinate Azolla roots, cell division appears to play a major role up to a certain root length, then increase in length is produced mainly by cell elongation.

Prior to the studies on the replicating patterns in bovine sex chromosomes, the frequency of cell division by the autoradiographic method of labeling with 3H-thymidine and the length of the cell cycle by the method of administration of 5–bro-modeoxyuridine (BrdU were examined in the cultured lymphocytes. The number of the cells labeled with 3H-thymidine increased punctually, and they almost attained their maximum number at 72 hours' labeling. In the cells to which BrdU had been added for over 36 hours, many cells passed through the cell cycle 3 times, and in the cells treated from 12 to 24 hours, almost all of the cells passed through the cell cycle two times. In the cells treated within 10 hours, almost none of the cells attained a full cell cycle. These results suggested that the length of the cell cycle in bovine cultured cells is about 12 hours. Untersuchungen zum Reproduktionsmuster der Geschlechtschromosomen des Rindes mit Hilfe von 5–Bromodeoxyuridin (BrdU) I. Die Häufigkeit der Zellteilung und die Länge des Zellzyklus Anhand des Reproduktionsmusters der Geschlechtschromosomen des Rindes wurde die Häufigkeit der Zellteilung mit Hilfe der autoradiographischen Methode durch Be-handlung mit H3 -Thymidin, und die Länge deszellzyklus durch Zugeben von 5–Bromodeoxyuridin (BrdU) in Lymphozytenkulturen untersucht. Die Zahl der mit H3--Thymidin behandelten Zellen stieg punktförmig an und erreichte ihre beinahe maximale Anzahl in 72 Stunden. Unter den Zellen, denen BrdU für 36 Stunden rugegeben war, gingen viele dreimal durch einen Zellzyklus; bei den Zellen, die 12–24 Stunden behandelt wurden, machten fast alle zweimal einen Zellzyklus durch. Von denjenigen Zellen, die nur 10 Stunden behandelt wurden, erreichte fast keine einen vollständigen Zellzyklus. Diese Ergebnisse zeigen, daβ die Länge eines Zellzyklus in Rinderzellkul-turen ungefähr 12 Stunden beträgt.

Breast cancer stem cells (BCSCs) are thought to be maintained in cancerous tissues by increased frequency of symmetric cell division at the expense of asymmetric cell divisionin the cancer tissues, although how this occurs it remain largely unknown. Here, we discovered that molecules interactinging with CasL 3 (MICAL3) contributed to tumor sphere formation in patient-derived breast cancer cells and tumor initiating activity in a patient-derived xenograft (PDX) model. Semaphorin (Sema)-stimulation induced complex formation involving MICAL3 and collapsin response mediator protein 2 (CRMP2), leading to stabilization of Numb in breast cancer cells. Neuropillin-1 (NP1), a receptor for Sema, and Numb were specifically expressed and colocalized at high levels in a BCSC-enriched population that induce tumor sphere formation. MICAL3 knockdown decreased symmetric cell division and increased asymmetric division of in NP1-high cancer stem-like cells that express high level of Numb at high levels. Thus, MICAL3/CRMP2/Numb pathway appears to play a critical role in the symmetric division of NP1-high cancer stem-like cells to expand the pool of BCSCs. Finally, we showed that breast cancer patients with higher expression levels of NP1 and Numb in cancer tissues show poor prognosis compared with those with lower expression levels of NP1 and Numb by using tissue microarray of breast cancer tissues. We provide a rationale for eradicating BCSC by blocking the symmetric division of BCSC by targeting molecules in Sema/NP1/MICAL3 pathway. Citation Format: Kana Tominaga, Masao Yano, Kei-ichiro Tada, Arinobu Tojo, Noriko Gotoh. Semaphorin signal via MICAL3 induces symmetrical cell division of cancer stem-like cells to confer aggressiveness in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5021. doi:10.1158/1538-7445.AM2017-5021

The INDETERMINATE DOMAIN Protein BROAD LEAF1 Limits Barley Leaf Width by Restricting Lateral Proliferation

Inhibitory effects of water soluble leaf leachates from Dittrichia viscosa on lettuce root growth, statocyte development and graviperception

BackgroundDespite the importance of the shoot apical meristem (SAM) in plant development and organ formation, our understanding of the molecular mechanisms controlling its function is limited. Genomic tools have the potential to unravel the molecular mysteries of the SAM, and legume systems are increasingly being used in plant-development studies owing to their unique characteristics such as nitrogen fixation, secondary metabolism, and pod development. Garden pea (Pisum sativum) is a well-established classic model species for genetics studies that has been used since the Mendel era. In addition, the availability of a plethora of developmental mutants makes pea an ideal crop legume for genomics studies. This study aims to utilise genomics tools in isolating genes that play potential roles in the regulation of SAM activity.ResultsIn order to identify genes that are differentially expressed in the SAM, we generated 2735 ESTs from three cDNA libraries derived from freshly micro-dissected SAMs from 10-day-old garden peas (Pisum sativum cv Torsdag). Custom-designed oligonucleotide arrays were used to compare the transcriptional profiles of pea SAMs and non-meristematic tissues. A total of 184 and 175 transcripts were significantly up- or down-regulated in the pea SAM, respectively. As expected, close to 61% of the transcripts down-regulated in the SAM were found in the public database, whereas sequences from the same source only comprised 12% of the genes that were expressed at higher levels in the SAM. This highlights the under-representation of transcripts from the meristematic tissues in the current public pea protein database, and demonstrates the utility of our SAM EST collection as an essential genetic resource for revealing further information on the regulation of this developmental process. In addition to unknowns, many of the up-regulated transcripts are known to encode products associated with cell division and proliferation, epigenetic regulation, auxin-mediated responses and microRNA regulation.ConclusionThe presented data provide a picture of the transcriptional profile of the pea SAM, and reveal possible roles of differentially expressed transcripts in meristem function and maintenance.

Lateral aerial organs are produced from the primordia initiated in the shoot apical meristem. Early stages of lateral organ growth require cell division and a sharp transition occurs late in development towards a cell expansion‐based growth. A current hypothesis indicates that cytokinin signalling in meristematic cells is downregulated to allow differentiation. Increased local auxin levels promoting growth accompany this event. The later stage coincides with jasmonic acid signalling required for flower maturarion. Within one organism, lateral organs of the same type can change in size and shape during ontogeny, a feature called heteroblasty. The developmental programme leading to final organ size may be modulated by environmental cues including photoperiod, temperature and abiotic stress. These environmental signals are integrated into the morphogenetic programme allowing better adaptation as a result of developmental plasticity. Key Concepts: Initial growth of lateral organs requires the downregulation of cytokinins allowing morphogenesis and differentiation. Early developmental stages are characterised by cell division, whereas later stages show a rapid transition to growth caused by cell expansion. There seems to be a compensation mechanism at the organ size level allowing increased cell expansion when cell division is compromised and vice versa, giving as a result lower organ size variation. A given species may develop lateral organs differing in size and shape during development, a phenomenon called heteroblasty. A coordination between a basic growth programme and the timing of organ formation seems to be responsible for this phenomenon. Plant organ size is the result of an underlying genetic programme and the interaction with environmental conditions that fine tune the final size of an organ.

Variation of spontaneous somatic pink mutation frequency was studied in the stamen hairs of Tradescantia KU 20 clone, a highly mutable blue/pink heterozygote. The spontaneous mutation frequency varied greatly from 4.03±1.21 to 120±7 and from 18.8±3.1 to 110±5 pink mutant events per 103 hairs when the plants were grown outdoors and in the greenhouse, respectively, being generally higher at lower temperature and also in the greenhouse than in outdoors. The spontaneous mutation frequency under controlled environmental conditions also varied from 3.06±0.37 to 40.8±3.1 pink mutant events per 103 hairs, showing a clearer negative correlation with temperature. It was found that the spontaneous mutation frequency under controlled environmental conditions increased when day/night temperature shifts were applied, especially with a 5°C shift than with 3°C shifts. The difference between the highest and the lowest mutation frequencies reached almost 40 times, and this clone was confirmed to be a temperature-sensitive mutable clone. A repair mechanism of DNA damages occurring spontaneously, which is more effective at higher temperature, thus presumably an enzymatic one, is very likely involved in the mutable nature of this clone.

Hormonal and cell division analyses in Watsonia lepida seedlings

The nucleosome assembly protein 1 (NAP1) is considered to be a conserved histone chaperone, facilitating the assembly of nucleosomes in all eukaryotes. However, studies in yeast and animal cells also indicated that NAP1 proteins have diverse functions likely independent of nucleosome-assembly activity. Here, we describe the isolation and characterization of cDNAs encoding NAP1-like proteins from the monocotyledon rice ( Oryza sativa L.) and the dicotyledon tobacco ( Nicotiana tabacum L.). Northern-blot analysis demonstrated that the two rice NAP1-like genes are predominantly expressed in stem tissues such as root and shoot apical meristems as well as in young flowers. During the cell cycle, all four tobacco NAP1-like genes are highly expressed, with one of them showing a slightly increased expression at the G1/S transition. These results are consistent with a role for plant NAP1-like proteins in cell division. In vitro binding assays revealed that different NAP1-like proteins bind, with distinct relative binding strengths, to different classes of histone. Intracellular localization analyses showed that some NAP1-like proteins could be targeted into the nucleus whereas others are exclusively cytoplasm-localized. It is thus likely that different plant NAP1-like proteins have distinct functions in vivo. Plant NAP1-like proteins were observed to concentrate around the metaphase plate and in the phragmoplast, suggesting a role in mitotic events and cytokinesis.

ABSTRACTCell proliferation is a key regulator of tissue morphogenesis. We examined cell proliferation and cell division in zebrafish lens epithelium by visualizing cell-cycle phases and nuclear positions, using fluorescent-labeled geminin and histone proteins. Proliferation was low in the anterior region of lens epithelium and higher in the marginal zone anterior to the equator, suggesting that the proliferation zone, called the germinative zone, is formed in zebrafish lens. Interestingly, cell-division orientation was biased longitudinally in the anterior region, shifted from longitudinal to circumferential along the anterior–posterior axis of lens sphere, and was biased circumferentially in the peripheral region. These data suggest that cell-division orientation is spatially regulated in zebrafish lens epithelium. The Hertwig rule indicates that cells tend to divide along their long axes. Orientation of long axes and cell division were biased similarly in zebrafish lens epithelium, suggesting that cell geometry correlates with cell-division orientation. A cell adhesion molecule, E-cadherin, is expressed in lens epithelium. In a zebrafish e-cadherin mutant, the long axes and cell-division orientation were shifted more longitudinally. These data suggest that E-cadherin is required for the spatial pattern of cell geometry and cell-division orientation in zebrafish lens epithelium.

Recent studies have shown that D-allose, a rare sugar, elicits antitumor effects on different types of solid cancers, such as hepatocellular carcinoma, non-small-cell lung cancer, and squamous cell carcinoma of the head and neck. In this study, we examined the effects of D-allose on the proliferation of human glioblastoma (GBM) cell lines (i.e., U251MG and U87MG) in vitro and in vivo and the underlying mechanisms. D-allose treatment inhibited the proliferation of U251MG and U87MG cells in a dose-dependent manner (3–50 mM). However, D-allose treatment did not affect cell cycles or apoptosis in these cells but significantly decreased the cell division frequency in both GBM cell lines. In a subcutaneous U87MG cell xenograft model, intraperitoneal injection of D-allose (100 mg/kg/day) significantly reduced the tumor volume in 28 days. These data indicate that D-allose-induced reduction in cell proliferation is associated with a subsequent decrease in the number of cell divisions, independent of cell-cycle arrest and apoptosis. Thus, D-allose could be an attractive additive to therapeutic strategies for GBM.