NMR metabolite profiling analysis reveals changes in phospholipid metabolism associated with the re-establishment of desiccation tolerance upon osmotic stress in germinated radicles of cucumber

The adaptation of metabolism is thought to play a role in the acquisition of desiccation tolerance (DT). However, the importance of such a role and whether specific regulatory pathways exist remain to be assessed. Using in vitro 31P and 13C nuclear magnetic resonance (NMR) spectroscopy and biochemical assays, we analysed metabolite profiles of perchloric extracts from germinating radicles of cucumber to identify changes in carbon and phosphate metabolism associated with DT. Emerged radicles measuring 2 mm long can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution with a water potential of 1.5 MPa. However, in 4-mm-long emerged radicles, this treatment was ineffective. This manipulable system enabled the discrimination of changes in metabolites associated with DT from those associated with the response to osmotic stress. Independent of radicle length, the PEG treatment resulted in an increase in sucrose (Suc) content, whereas glucose (Glc), fructose (Fru) and the hexose phosphate pool, as well as phosphoenolpyruvate decreased three- to fourfold. In addition, three derivatives arising early during phospholipid catabolism (glycerylphosphorylcholine, glycerylphosphorylethanolamine and glycerylphosphorylinositol) appeared in the PEG-treated radicles. Interestingly, phospholipid degradation was much more pronounced in osmotically challenged radicles that remain sensitive to drying. This was proved by the appearance of catabolites, such as phosphocholine and phosphoethanolamine, solely in 4 mm PEG-treated radicles. Furthermore, glycerol-3-phosphate and its derivative 3-phosphoglycerate increased significantly. Our data suggest that the metabolic response leading to the re-establishment of DT is not entirely identical to that of an osmotic response. It is inferred that membrane remodelling and/or increased phospholipid catabolism is an adaptive response common to osmotic adjustment and DT but is controlled differently in tolerant and sensitive radicles.     

Cell division and subsequent radicle protrusion in tomato seeds are inhibited by osmotic stress but DNA synthesis and formation of microtubular cytoskeleton are not

We studied cell cycle events in embryos of tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds during imbibition in water and during osmoconditioning ("priming") using both quantitative and cytological analysis of DNA synthesis and beta-tubulin accumulation. Most embryonic nuclei of dry, untreated control seeds were arrested in the G(1) phase of the cell cycle. This indicated the absence of DNA synthesis (the S-phase), as confirmed by the absence of bromodeoxyuridine incorporation. In addition, beta-tubulin was not detected on western blots and microtubules were not present. During imbibition in water, DNA synthesis was activated in the radicle tip and then spread toward the cotyledons, resulting in an increase in the number of nuclei in G(2). Concomitantly, beta-tubulin accumulated and was assembled into microtubular cytoskeleton networks. Both of these cell cycle events preceded cell expansion and division and subsequent growth of the radicle through the seed coat. The activation of DNA synthesis and the formation of microtubular cytoskeleton networks were also observed throughout the embryo when seeds were osmoconditioned. However, this pre-activation of the cell cycle appeared to become arrested in the G(2) phase since no mitosis was observed. The pre-activation of cell cycle events in osmoconditioned seeds appeared to be correlated with enhanced germination performance during re-imbibition in water.     

Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism

Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of IFs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF-MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of IFs with MTs, as assayed by the collapse of IFs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of IFs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT-IF interaction was mapped by microinjecting tubulin fragments of alpha-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (alpha-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of alpha-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and alpha-C Glu) that disrupted the IF-Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of IFs to MTs via a mechanism that is likely to involve kinesin.     

β-Tubulin accumulation and DNA synthesis are sequentially resumed in embryo organs of cucumber (Cucumis sativus L.) seeds during germination

Summary Resumption of DNA synthetic activities and -tubulin accumulation was studied in embryo organs of germinating cucumber (Cucumis sativus L.) seeds. Flow-cytometric analysis indicated the existence of 2C, 4C, and 8C nuclei in the radicle of mature embryos, whereas in cotyledons most of the cells contained nuclei with 2C DNA content. Upon imbibition of water, nuclear DNA replication was initiated in the radicle within 15 h, subsequently spreading towards the cotyledons. Bromodeoxyuridine incorporation preceded detectable changes in the relative amounts of DNA, implying the occurrence of putative DNA repair. Organellar DNA synthesis occurred independently of the nuclear DNA synthetic cycle. Western blotting and immunohistochemical localization demonstrated that the constitutive level of -tubulin originated from preserved -tubulin granules. During imbibition, disappearance of fluorescent tubulin granules, accumulation of -tubulin, and formation of microtubular cytoskeleton were found in the radicle, but not in the cotyledon areas. Mitosis only occurred after radicle protrusion at 21 h of imbibition. It is concluded that the differences in the initiation and progress of these cellular and molecular events are associated with the discrete behaviors of the radicle and the cotyledons upon imbibition. The formation of cortical microtubular cytoskeleton and the accumulation of tubulins are important features in preparation of radicle protrusion, whereas DNA synthesis may contribute to postgerminative growth.     

Changes in endogenous cytokinins of celery (Apium graveolens L.) seeds following an osmotic priming or growth regulator soak treatment

Celery seeds were less thermoinhibited when dried back after a seed soak treatment with the gibberellins A₄ and A₇ (GA_4/7) plus ethephon (G+E) or an osmotic priming treatment in the light with polyethylene glycol (PEG). At temperatures between 18 and 25° in the dark, 50 percent of the PEG-treated seeds germinated after 3 days whereas G+E-treated seeds required 7 days and untreated seeds did not germinate at all.Irrespective of treatment, dry control and dried-back, treated seeds contained very little detectable cytokinin activity. However, when such seeds were imbibed for 18 h in the dark and then analysed immediately with the soybean callus bioassay, less cytokinin activity was detected in both G+E and PEG seeds than in the untreated seeds. In particular, cytokinins with the HPLC properties of zeatin and its riboside were decreased in G+E seeds and virtually absent from PEG seeds. Conversely, extracts from PEG and to a lesser extent G+E seeds contained activity which chromatographically resembled cytokinin glucosides whereas this was absent from untreated seeds.     

The role of sugars and hexose phosphorylation in regulating the re-establishment of desiccation tolerance in germinated radicles of Cucumis sativa and Medicago truncatula

This study examined whether sugars and hexose phosphorylation participate in the regulatory mechanisms that induce desiccation tolerance (DT) in seeds. In germinated desiccation-sensitive radicles of Cucumis sativa and Medicago truncatula , DT was re-established by an osmotic treatment using polyethylene glycol (PEG) for several days. In cucumber, Glc kinase activity (EC 2.7.1.1) transiently peaked early during PEG incubation before the induction of DT in protruded radicles, whereas Fru kinase activity (EC 2.7.1.4) increased progressively during the re-establishment of DT. Glucosamine (GAM, a competitive inhibitor of HXK) was able to repress the PEG-induced DT in both species, whereas hexose and poorly metabolizable hexose analogues had no effect. GAM was effective in repressing DT only early during PEG incubation, indicating that this effect is transient. Both Glc and Man fully rescued GAM-inhibited DT. PEG-induced accumulation of Suc was not affected by GAM. Isocitrate lyase (ICL) gene expression, which is known to be regulated by hexoses, responded to the re-establishment of DT and GAM feeding. In cucumber, expression of ICL was repressed after 6 h of PEG incubation whereas GAM feeding led to ICL de-repression. When GAM could no longer inhibit the re-establishment of DT, neither were steady-state levels of ICL influenced. The implication of HXK as a catalytic regulator and sugar-sensor in DT is discussed.     

Non-geotropic Growth Curvatures in the Striga Radicle

When seedlings of the root parasite Striga asiatica Benth. (=S. lutea Lour.) are grown in0 the absence of the host the radiclegrows to a length of about 4 mm. On emergence the radicle showsa strong curvature which is brought about by a failure of cellextension on one side. The inhibited cells generally remainun-extended throughout the life of the seedling. Neither thesodium salt of indole-3-acetic acid nor Inhibitor-ß(which is present in the seeds), induced permanent inhibitionsto the radicle cells when applied externally, as did the endogenouscurvature factor. 2: 3: 5-tri-iodobenzoic acid reduced curvatureby promoting extension of the normally inhibited cells.     

Changes in tubulin protein expression in guard cells of Vicia faba L. accompanied with dynamic organization of microtubules during the diurnal cycle

We previously reported that the organization of microtubules (MTs) in guard cells of Vicia faba L. shows dynamic diurnal changes [Fukuda et al. (1998) Plant Cell Physiol. 39: 80]. Here, we report a method to directly extract total proteins from guard cells to investigate the biochemical changes in guard cells of Vicia faba L. during the diurnal cycle. Electrophoretic profiles of total proteins of guard cells showed distinct patterns with the time of extraction. Immunoblot analysis also demonstrated changes in alpha-tubulin and beta-tubulin contents with the diurnal cycle. Both tubulins were abundant at 6:00 h and 12:00 h but were almost undetectable at 24:00 h. Although treatment with either actinomycin D or cycloheximide at 18:00 h inhibited neither radial organization of cortical MTs nor stomatal opening, that at 6:00 h inhibited both. These results suggest that the dynamic diurnal changes in the organization of MTs in guard cells and stomatal movement of Vicia faba L. may be, at least partly, regulated by de novo synthesis and decomposition of tubulin molecules in guard cells.     

Dehydration Injury in Germinating Soybean (Glycine max L. Merr.) Seeds

The sensitivity of soybean (Glycine max L. Merr. cv Maple Arrow) seeds to dehydration changed during germination. Seeds were tolerant of dehydration to 10% moisture if dried at 6 hours of imbibition, but were susceptible to dehydration injury if dried at 36 hours of imbibition. Dehydration injury appeared as loss of germination, slower growth rates of isolated axes, hypocotyl and root curling, and altered membrane permeability. Increased electrolyte leakage due to dehydration treatment was observed only from isolated axes but not from cotyledons, suggesting that cotyledons are more tolerant of dehydration. The transition from a dehydration-tolerant to a dehydration-susceptible state coincided with radicle elongation. However, the prevention of cell elongation by osmotic treatment in polyethylene glycol (−6 bars) or imbibition in 20 micrograms per milliliter cycloheximide did not prevent the loss of dehydration tolerance suggesting that neither cell elongation nor cytoplasmic protein synthesis was responsible for the change in sensitivity of soybean seeds to dehydration. Furthermore, the rate of dehydration or rate of rehydration did not alter the response to the dehydration stress.     

Cell division versus cell elongation: the control of radicle elongation during thermoinhibition of Tagetes minuta achenes

Endogenous embryo factors, which act mainly in the radicle, prevent germination in Tagetes minuta at high temperatures. These factors act to prevent cell elongation, which is critical for radicle protrusion under optimal conditions. Once the radicle has emerged both cell elongation and cell division are required for post-germination growth. Germination can be induced at high temperatures by fusicoccin, which rapidly stimulates cell elongation. In addition, priming seeds at 25 °C on polyethylene glycol (PEG) 6000 and mannitol could also induce germination on water at 36 °C, indicating that priming prevents radicle protrusion at a point subsequent to the point of control in thermoinhibited achenes. Flow cytometry studies revealed that DNA synthesis occurs during thermoinhibition and the inhibition of DNA synthesis during this process inhibits subsequent germination on water under optimal conditions, suggesting a protective role for DNA synthesis in thermoinhibited achenes of T. minuta.     

Calmodulin colocalization with cold-stable and nocodazole-stable microtubules in living PtK1 cells

To investigate the association of calmodulin (CaM) with microtubules (MTs) in the mitotic apparatus (MA), the distributions of both CaM and tubulin were examined in mitotic PtK1 cells in which MT subclasses had been selectively removed or altered by treatment with cold or with the MT inhibitor, nocodazole. A fluorescent CaM conjugate with tetramethylrhodamine isothiocyanate (CaM-TRITC) was microinjected into living cells, and the CaM distribution in the living cell was compared to the distribution of MTs indicated by tubulin immunofluorescence. In cells which had been treated for 2 h at 0 to 4 degrees C or with a low (0.03 micrograms/ml) dose of nocodazole, the only MTs remaining appeared to be kinetochore MTs (kMTs). The distribution of microinjected CaM-TRITC in these cells was indistinguishable from that found in untreated cells and appeared to be colocalized with the kMTs. In cells which were treated with a high (3.0 micrograms/ml) dose of nocodazole, only short MTs remained. When CaM-TRITC was injected into these cells, it formed a somewhat punctate distribution near the chromosomes and, after tubulin immunofluorescence processing, colocalized with what appeared to be remnants of kMTs. We believe that these observations support the hypothesis that CaM exists in the MA in a structural association with kMTs.     

Protein Synthesis in the Axes of Polyethylene Glycol-Treated Pea Seed and during Subsequent Germination

Germination of Alaska pea seeds is inhibited by –0.3 MPapolyethylene glycol but upon subsequent transfer to water, germinationis completed rapidly and radicle emergence occurs more quicklythan in water-imbibed seeds. Protein synthesis is reduced inthe axes of seeds imbibed on PEG but increases upon their returnto water, though not to the level exhibited by axes germinatedon water. Mobilization of proteins in the axes is retarded bytheir failure to complete germination on PEG, although somedoes occur. The quantitative reduction in protein synthesisresulting from incubation in osmoticum is not accompanied bymarked qualitative changes. The block to germination is notobviously associated with a restriction in synthesis of anyparticular protein or set of proteins; conversely, no ‘water-stress’proteins are synthesized in the presence of PEG. The synthesisof growth-specific proteins is prevented by PEG, but these increaseupon relief from the osmoconditioning treatments. These observationsdispute earlier claims for accelerated protein synthesis resultingfrom PEG treatments. Key words: Osmotic priming, Pisum sativum, germination, protein synthesis     

Amounts of Nuclear DNA and Internal Morphology of Gibberellin- and Abscisic Acid-deficient Tomato (Lycopersicon esculentum Mill.) Seeds During Maturation, Imbibition and Germination

Using X-ray photography and flow cytometry, the internal morphologyand DNA replication activity of wild type (wt), GA- (gib-1 )and ABA-deficient (sit^w ) tomato (Lycopersicon esculentum Mill.cv. Moneymaker) mutant seeds were studied. During seed formation,from 30 to 45 d after pollination (DAP) the endosperm becomessolid and the seed starts to gain desiccation tolerance. Atthis time significant changes occur in the amounts of DNA inradicle tip cells. At 30 DAP, radicle tip cells of the threegenotypes manifest about 60% of 2C, 30% of 4C and 10% of 8Camounts of DNA. Upon maturation (45 DAP onwards), most cellsin the seeds of the three genotypes arrest in the G₁phase ofthe cell-cycle with 2C amounts of DNA. However, a relativelyhigh proportion of cells with 4C amounts of DNA was detectedin the radicle tip cells ofsit^w compared with wild type andgib-1. At the well-matured stage (60 DAP), there were about 2% ofseeds with free space in wild type andgib-1 , and about 13%insit^w . At the over-matured stage (75 DAP), even more seedswith free space were found insit^w , whereas no increase in theproportion of the seeds with free space was detected in theother two genotypes. In -1.0 MPa PEG-6000 with or without 10µM GA₄₊₇, no germination occurred in well-matured wildtype andgib-1 seeds, whether or not they were dried after harvest.However,sit^w seeds were able to germinate both in over-maturefruit and in -1.0 MPa PEG-6000. Priming of dried seeds in -1.0MPa PEG induced a large amount of free space in almost all seedsof the three genotypes, and nuclear DNA synthesis in the radicletip cells of wild type andsit^w seeds. However, PEG priming offresh (non-dried) seeds had no effect on the amount of freespace and 2C/4C DNA ratios in wild type orgib-1 seeds, but didinduce free space in about 20–25% ofsit^w seeds and provoked4C signals insit^w seeds. Removal of the endosperm and testaopposite the radicle tip of seeds resulted in root protrusion,the induction of free space and an increase of 4C DNA signalsin the three genotypes. It is concluded that ABA is crucialfor the efficient arrest of tomato embryo radicle tip cellsin G₁phase upon maturation, whereas GAs play an important rolein re-initiating 4C DNA levels upon germination. Dormancy; flow cytometry; free space; Lycopersicon esculentum ; maturation; priming; seed; tomato     

渗透胁迫对黑麦幼苗活性氧和抗氧化酶活性的影响

用20%聚乙二醇(PEG 6000)研究了渗透胁迫对黑麦(Secale cereale L.)幼苗活性氧(reactive oxygen species, ROS)和主要抗氧化酶—— 超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)、抗坏血酸过氧化物酶(ascorbate peroxidase, APX)和谷胱甘肽还原酶(glutathione reductase, GR)活性的影响。结果表明, 与对照相比, PEG处理明显提高了叶子和根中丙二醛(malondialdehyde, MDA)的含量、ROS的水平和以上4种抗氧化酶的活性。渗透胁迫下,叶子和根中MDA和ROS水平变化的规律基本相似, 但抗氧化酶活性在2种器官中表现不完全相同, 叶子中CAT的活性在对照和处理中无显著差异, 但在根中差异明显, 表明叶子中SOD、APX和GR在植物应答渗透胁迫中起重要作用, 而根中这4种抗氧化酶都参与植物对胁迫的反应。GR活性随PEG处理变化幅度显著高于其它抗氧化酶, 表明GR在黑麦应答渗透胁迫中所起作用可能强于其它抗氧化酶。     

Lack of Inhibiting Effects of Abscisic Acid on Seeds of Haplopappus gracilis (Nutt.) Gray Pregerminated in Water for Short Times

Abscisic acid (ABA) inhibits germination of Haplopappus gracilisseeds. If seeds are germinated in water until radicle protrusionand then transferred to ABA, no inhibiting effect is observedand growth goes on normally. The same behaviour is observedif embryos (i.e. seeds deprived of coats) are pregerminatedin water for 12 h. No growth substance (cytokinins or gibberellins) is able toreverse in short times the inhibiting effects of ABA on germinationand on resumption of DNA synthesis; hence it is unlikely thatsome growth regulator synthesized during the early hours ofgermination can account for a subsequent neutralization of theinhibiting effects of ABA. It is suggested that ABA is metabolized to some compound devoidof hormonal activity in the seeds during the incubation in water.     

Changes in Some ATP-Dependent Activities in Seeds during Treatment with Polyethyleneglycol and during the Redrying Process

During the imbibition of seeds in polyethyleneglycol (PEG),increasing amounts of ATP accumulated up to 24 h. Similar amountsaccumulated in the seeds during 4–5 h of imbibition inwater. Radioactive amino acids were increasingly incorporatedin the acid-insoluble fraction up to 24 h imbibition in PEG,as well as in water, after which a sharp decrease occurred upto 5 d of imbibition. If seeds were imbibed in PEG or waterin the presence of radioactive acetate, water-insoluble radioactivityincreased linearly in seeds during 5 d of imbibition. The amountsof incorporated amino acids or acetate were about double inPEG-imbibed as compared with in water-imbibed seed. The incorporationof AMP into the acid-insoluble fraction in seeds imbibed inPEG in the presence of radioactive AMP levelled off after 24h followed by a sharp decrease of up to 10% of the peak 5 dafter the start of imbibition. In water-imbibed seeds the incorporationof AMP continued to increase during at least 5 d of imbibition.During redrying of PEG-treated seeds (24 h), at least 80% ofthe accumulated ATP decreased during 18 d. The total radioactiveamino acids and nucleotide decreased during 3 d of redryingby 20% and 60%, respectively. At that time, the acid-insolubleincorporates increased by 20% and 50%, respectively. Some ofthe AMP was released as CO₂. Key words: AMP, Germination, Nucleic acid synthesis, Osmoconditioning, PEG, Protein synthesis     

Induction by gamma-radiation of DNA synthesis in radicle cells of germinating seeds of Pisum sativum l.

In radicle meristem cells of germinating seeds of the pea (Pisum sativum L) before the onset of replicative synthesis of DNA, irradiation with 2-3 krad of gamma-rays induced the incorporation of 3H-thymidine (3H-TdR). Maximum isotope incorporation was noted during the first 2 hours after irradiation. Higher doses of radiation suppressed 3H-TdR incorporation. It was not seen after gamma-irradiation of air-dried seeds, nor after fast-neutron irradiation. The replication inhibitors hydroxyurea and 5-aminouracil had no effect on the gamma-induced incorporation of 3H-TdR, Whereas caffeine and acriflavine inhibited it to some extent. It is suggested that the gamma-radiation-induced incorporation of 3H-TdR in meristem cells during the pre-replicative period may be connected with repair phenomena.     

Stabilization of Cortical Microtubules in Maize Mesocotyl Cells by Gibberellin A3

Effects of GA₃ on the stability of cortical microtubules (MTs)were studied in mesocotyl cells of etiolated maize seedlings.Propyzamide, an MT-disrupting agent specific for plant tubulin,disrupted cortical MTs in cells in the upper regions of mesocotylsof GA₃-untreated seedlings and caused swelling of the cells.GA₃ prevented propyzamide from disrupting MTs and from causingsuch swelling. Chilling of mesocotyls at 4°C for 60 minbrought about the disruption of cortical MTs in cells in theupper regions of mesocotyls of GA₃-untreated seedlings, butnot in cells in corresponding regions of GA₃-treated seedlings,suggesting that treatment with GA₃ increased the stability ofcortical MTs in maize mesocotyl cells. Cortical MTs in protoplastsisolated from mesocotyls of GA₃-untreated seedlings failed towithstand chilling at 0°C for 90 min, while those in protoplastsisolated from mesocotyls of GA₃-treated seedlings withstoodchilling successfully. It appears that the cell wall is notinvolved in the stabilization of cortical MTs by GA₃ in maizemesocotyl cells. (Received July 6, 1993; Accepted November 29, 1993)     

Taxol inhibits stimulation of cell DNA synthesis by human cytomegalovirus

The microtubule (MT)-stabilizing drug, taxol, inhibited human cytomegalovirus (CMV)-initiated cell DNA synthesis by up to 100% in serum-arrested mouse embryo (ME) fibroblasts that were abortively infected by CMV. Taxol concentrations known to increase MT polymerization and to stabilize existing MTs (10 to 20 micrograms/ml) blocked CMV-stimulated cell DNA synthesis, while taxol concentrations of 2.5 micrograms/ml, or less, did not. Taxol maximally inhibited CMV initiation of cell DNA synthesis when added 3 h after virus infection and inhibited this initiation by greater than 50% when added up to 12 h after CMV infection. Control experiments suggest that taxol specifically inhibited CMV-stimulated cell DNA synthesis. Pretreatment of CMV stock with taxol did not reduce the stimulatory effect of CMV on cell DNA synthesis and taxol had no detectable effect on CMV-specific early protein synthesis. Moreover, taxol did not appear to alter thymidine pool sizes, affect cell viability, or compromise the DNA synthetic machinery in CMV-infected cells. Since taxol increases tubulin polymerization and inhibits MT disassembly, these results suggest that dynamic changes in MTs or in the pool of free tubulin subunits are necessary for CMV to stimulate cell entry into a proliferative cycle.     

The abundance of alpha-tubulin mRNA increases during ciliary regeneration in Tetrahymena, and this occurs independently of the soluble tubulin content

Control mechanisms of tubulin synthesis are analyzed during ciliary regeneration of the ciliate Tetrahymena. Titration of the alpha-tubulin mRNA concentrations during the regeneration period reveal that enhancement of tubulin synthesis is preceded and accompanied by increased concentrations of tubulin mRNA molecules. Stimulation of tubulin synthesis is independent of the pool size of soluble tubulin molecules, as suggested by at least two independent lines of evidence: First, like cells of normal phenotype a temperature sensitive size mutant enhances tubulin synthesis as well as tubulin mRNA concentration during ciliary regeneration, although these large mutant cells have a much higher concentration and amount of soluble tubulin molecules in the cytoplasm. Second, slowly regenerating cells of normal phenotype shift-up their concentration of tubulin mRNA molecules already before a time, when ciliary outgrowth might cause a significant depletion of the pool of soluble tubulin molecules. Thus, neither an induction of tubulin synthesis nor an increase in tubulin mRNA molecules is mediated via changes in the pool size of soluble tubulin molecules.