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PubMed2000

Kinetic analysis of a molecular model of the budding yeast cell cycle.

The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1-3 and Clb1-6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling "Start" (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and "Finish" (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast.

Cell Cycle↗

PubMed2003

A Bni4-Glc7 phosphatase complex that recruits chitin synthase to the site of bud emergence.

Bni4 is a scaffold protein in the yeast Saccharomyces cerevisiae that tethers chitin synthase III to the bud neck by interacting with septin neck filaments and with Chs4, a regulatory subunit of chitin synthase III. We show herein that Bni4 is also a limiting determinant for the targeting of the type 1 serine/threonine phosphatase (Glc7) to the bud neck. Yeast cells containing a Bni4 variant that fails to associate with Glc7 fail to tether Chs4 to the neck, due in part to the failure of Bni4(V831A/F833A) to localize properly. Conversely, the Glc7-129 mutant protein fails to bind Bni4 properly and glc7-129 mutants exhibit reduced levels of Bni4 at the bud neck. Bni4 is phosphorylated in a cell cycle-dependent manner and Bni4(V831A/F833A) is both hyperphosphorylated and mislocalized in vivo. Yeast cells lacking the protein kinase Hsl1 exhibit increased levels of Bni4-GFP at the bud neck. GFP-Chs4 does not accumulate at the incipient bud site in either a bni4::TRP1 or a bni4(V831A/F833A) mutant but does mobilize to the neck at cytokinesis. Together, these results indicate that the formation of the Bni4-Glc7 complex is required for localization to the site of bud emergence and for subsequent targeting of chitin synthase.

Cell Division↗

PubMed2005

Role of the septin ring in the asymmetric localization of proteins at the mother-bud neck in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, septins form a scaffold in the shape of a ring at the future budding site that rearranges into a collar at the mother-bud neck. Many proteins bind asymmetrically to the septin collar. We found that the protein Bni4-CFP was located on the exterior of the septin ring before budding and on the mother side of the collar after budding, whereas the protein kinase Kcc4-YFP was located on the interior of the septin ring before budding and moved into the bud during the formation of the septin collar. Unbudded cells treated with the actin inhibitor latrunculin-A assembled cortical caps of septins on which Bni4-CFP and Kcc4-YFP colocalized. Bni4-CFP and Kcc4-YFP also colocalized on cortical caps of septins found in strains deleted for the genes encoding the GTPase activating proteins of Cdc42 (RGA1, RGA2, and BEM3). However, Bni4-CFP and Kcc4-YFP were still partially separated in mutants (gin4, elm1, cla4, and cdc3-1) in which septin morphology was severely disrupted in other ways. These observations provide clues to the mechanisms for the asymmetric localization of septin-associated proteins.

Actins↗

PubMed2005

Bud composition, branching patterns and leaf phenology in cerrado woody species.

BACKGROUND AND AIMS: Plants have complex mechanisms of aerial biomass exposition, which depend on bud composition, the period of the year in which shoot extension occurs, branching pattern, foliage persistence, herbivory and environmental conditions. METHODS: The influence of water availability and temperature on shoot growth, the bud composition, the leaf phenology, and the relationship between partial leaf fall and branching were evaluated over 3 years in Cerrado woody species Bauhinia rufa (BR), Leandra lacunosa (LL) and Miconia albicans (MA). KEY RESULTS: Deciduous BR preformed organs in buds and leaves flush synchronously at the transition from the dry to the wet season. The expansion time of leaves is <1 month. Main shoots (first-order axis, A1 shoots) extended over 30 d and they did not branch. BR budding and foliage unfolds were brought about independently of inter-annual rainfall variations. By contrast, in LL and MA evergreen species, the shoot extension rate and the neoformation of aerial organs depended on rainfall. Leaf emergence was continuous for 2-6 months and lamina expansion took place over 1-4 months. The leaf life span was 5-20 months and the main A1 shoot extension happened over 122-177 d. Both evergreen species allocated biomass to shoots, leaves or flowers continuously during the year, branching in the middle of the wet season to form second-order (A2 shoots) and third-order (A3 shoots) axis in LL and A2 shoots in MA. Partial shed of A1 shoot leaves would facilitate a higher branching intensity A2 shoot production in LL than in MA. MA presented a longer leaf life span, produced a lower percentage of A2 shoots but had a higher meristem persistence on A1 and A2 shoots than LL. CONCLUSIONS: It was possible to identify different patterns of aerial growth in Cerrado woody species defined by shoot-linked traits such as branching pattern, bud composition, meristem persistence and leaf phenology. These related traits must be considered over and above leaf deciduousness for searching functional guilds in a Cerrado woody community. For the first time a relationship between bud composition, shoot growth and leaf production pattern is found in savanna woody plants.

Bauhinia↗

PubMed2003

Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate bud cortex interactions.

The budding yeast spindle aligns along the mother- bud axis through interactions between cytoplasmic microtubules (CMs) and the cell cortex. Kar9, in complex with the EB1-related protein Bim1, mediates contacts of CMs with the cortex of the daughter cell, the bud. Here we established a novel series of events that target Kar9 to the bud cortex. First, Kar9 binds to spindle pole bodies (SPBs) in G(1) of the cell cycle. Secondly, in G(1)/S the yeast Cdk1, Cdc28, associates with SPBs and phosphorylates Kar9. Thirdly, Kar9 and Cdc28 then move from the SPB to the plus end of CMs directed towards the bud. This movement is dependent upon the microtubule motor protein Kip2. Cdc28 activity is required to concentrate Kar9 at the plus end of CMs and hence to establish contacts with the bud cortex. The Cdc28-regulated localization of Kar9 is therefore an integral part of the program that aligns spindles.

CDC2 Protein Kinase↗

PubMed1998

Serine protease inhibitors expressed in the process of budding of tunicates as revealed by EST analysis.

To identify genes expressed during budding of the tunicate Polyandrocarpa misakiensis, we isolated and sequenced 624 clones from a directionally constructed cDNA library to prepare a catalog of expressed sequence tags (ESTs). A total of 233 ESTs matched genes of known sequence in the SwissProt database. About 24% out of them showed high similarity to ribosomal proteins, twice the value (12%) of pre-budding animals. ESTs involved in the respiratory chain also appeared with significant redundancy, suggesting that tunicate budding is accompanied by the enhancement of energy conversion as well as protein synthesis. Serine protease inhibitor (serpin) afforded another striking example of a gene that was highly expressed in the process of budding. The deduced amino acid sequences of five serpin cDNAs all had two consensus signatures of the Kazal's type of secretory protease inhibitor, one of which had an active site for trypsin and the other for elastase. In line with this, recombinant GST-fusion protein showed both trypsin and elastase inhibitor activities. In accordance with the EST analysis, the hemolymph taken from the budding stage showed the highest activity of trypsin inhibitor. We discuss a possible role that Polyandrocarpa serpins may play in bud development by counteracting trypsin-like serine protease, which could facilitate dedifferentiation of formative tissues.

Amino Acid Sequence↗

PubMed2000

Acropetal disappearance of PsAD1 protein in pea axillary buds after the release of apical dominance.

We recently isolated PsAD1 cDNA from pea (Pisum sativum L. cv. Alaska) seedlings, whose mRNA abundantly accumulated in dormant axillary buds and disappeared after decapitation [Madoka and Mori (2000) Plant Cell Physiol. 41: 274]. To further elucidate the function of PsAD1, we investigated the temporal and spatial distribution patterns of PsAD1 protein using Western blot and immunocytochemical analyses. Western blot analyses showed that accumulation patterns of PsAD1 protein in axillary buds after decapitation and in response to IAA and 6-benzyladenine were the same as those of PsAD1 mRNA. Immunocytochemical analyses showed that (1) PsAD1 proteins were localized in the procambia, leaf primordia, apical meristem, and secondary axillary buds in the dormant axillary bud, and this distribution was the same as that of PsAD1 mRNA, (2) PsAD1 proteins acropetally disappeared after decapitation, and (3) the growth of axillary buds occurred in the same manner. These acropetal changes occur in a manner similar to the way in which the procambium differentiates into vascular tissue. These results suggest that PsAD1 plays some role in the inhibition of growth and differentiation, or in the maintenance of the dormant state in axillary buds.

Amino Acid Sequence↗

PubMed2005

Suppression of tiller bud activity in tillering dwarf mutants of rice.

In this study, we analyzed five tillering dwarf mutants that exhibit reduction of plant stature and an increase in tiller numbers. We show that, in the mutants, axillary meristems are normally established but the suppression of tiller bud activity is weakened. The phenotypes of tillering dwarf mutants suggest that they play roles in the control of tiller bud dormancy to suppress bud activity. However, tillering dwarf mutants show the dependence of both node position and planting density on their growth, which implies that the functions of tillering dwarf genes are independent of the developmental and environmental control of bud activity. Map-based cloning of the D3 gene revealed that it encodes an F-box leucine-trich repeat (LRR) protein orthologous to Arabidopsis MAX2/ORE9. This indicates the conservation of mechanisms controlling axillary bud activity between monocots and eudicots. We suggest that tillering dwarf mutants are suitable for the study of bud activity control in rice and believe that future molecular and genetic studies using them may enable significant progress in understanding the control of tillering and shoot branching.

Amino Acid Sequence↗

PubMed1995

Dormancy release and chilling requirement of buds of latitudinal ecotypes of Betula pendula and B. pubescens.

Bud burst and dormancy release of latitudinal ecotypes of Betula pendula Roth and B. pubescens Ehrh. from Denmark ( approximately 56 degrees N), mid-Norway ( approximately 64 degrees N) and northern Norway ( approximately 69 degrees N) were studied in controlled environments. Dormant seedlings were chilled at 0, 5 or 10 degrees C from October 4 onward and then, at monthly intervals from mid-November to February, batches of seedlings were held at 15 degrees C in an 8-h (SD) or 24-h (LD) photoperiod to permit flushing. A decline in days to bud burst occurred with increasing chilling time in all ecotypes. In November, after 44 chilling days, time to bud burst was least in plants chilled at 0 and 5 degrees C. The difference diminished with increasing chilling time, and in February, after 136 chilling days, bud burst was earliest in plants chilled at 10 degrees C. Long photoperiods during flushing significantly reduced thermal time after short chilling periods (44 and 74 days), but had no effect when the chilling requirement was fully met after 105 or more chilling days. No significant difference in these responses was found between the two species. In both species, chilling requirement decreased significantly with increasing latitude of origin. Bud burst was normal in seedlings overwintered at 12 degrees C, but was erratic and delayed in seedlings overwintered at 15 and especially at 21 degrees C, indicating that the critical overwintering temperature is between 12 and 15 degrees C. We conclude that there is little risk of a chilling deficit in birch under Scandinavian winter conditions even with a climatic warming of 7-8 degrees C. The likely effects of a climatic warming include earlier bud burst, a longer growing season and increased risk of spring frost injury, especially in high latitude ecotypes.

Journal Article↗

PubMed1998

Effects of photoperiod and temperature on the timing of bud burst in Norway spruce (Picea abies).

We examined the effects of several photoperiod and temperature regimes imposed during the winter-spring period on the timing of bud burst in rooted cuttings of Norway spruce (Picea abies (L.) Karst.) grown in a greenhouse in Finland. The treatments were initiated in November and December after the cuttings had been exposed to natural chilling and freezing events. Irrespective of the treatments applied, time to bud burst decreased with increased duration of previous exposure to natural chilling and freezing events. Fluctuating day/night temperatures and continuous lengthening of the photoperiod hastened bud burst. Shortening the photoperiod delayed bud burst, suggesting that little or no ontogenetic development toward bud burst takes place during mild periods before the winter solstice. In the case of climatic warming, this phenomenon may prevent the premature onset of growth that has been predicted by computer simulations with models that only consider temperature regulation of bud burst.

Journal Article↗

PubMed1998

Freezing behaviors in leaf buds of cold-hardy conifers visualized by NMR microscopy.

1H-Nuclear magnetic resonance (NMR) microscopy was used to study freezing behavior in wintering leaf buds of Momi fir (Abies firma Sieb. et Zucc.) and Japanese red pine (Pinus densiflora Sieb. et Zucc.). The images acquired predominantly reflected the density of mobile (i.e., non-ice) protons mainly from unfrozen water. By comparing images taken at various subfreezing temperatures, we determined which tissues produced the high and low temperature exotherms detected by differential thermal analyses. Typical extra-organ freezing was successfully imaged in leaf buds of A. firma. The bud scales readily froze at -7 degrees C, but shoot primordia remained supercooled to -14 degrees C in December buds and to -21 degrees C in March buds. The size of supercooled shoot primordia was reduced with decreasing temperature, indicating a gradual decrease in water content of the shoot primordia. In contrast, the signal from shoot primordia of P. densiflora disappeared between -7 and -14 degrees C, corresponding to the high temperature exotherm at -8 degrees C, indicating extracellular freezing of the shoot primordia. The xylem and bark tissues readily froze at -7 degrees C in A. firma and between -7 and -14 degrees C in P. densiflora. We conclude that NMR microscopy can noninvasively provide more spatially specific information about freezing behavior in leaf buds than traditional methods such as differential thermal analysis. In particular, it allows the organized and harmonized freezing behaviors in complex organs to be visualized directly thereby revealing the diversity of mechanisms involved in freezing behaviors.

Journal Article↗

PubMed2000

Influence of temperature on the dynamics of ATP, ADP and non-adenylic triphosphate nucleotides in vegetative and floral peach buds during dormancy.

The nucleotides test of endodormancy, which is based on the capacity of tissues to synthesize ATP and non-adenylic triphosphate nucleotides (NTP), cannot be used for floral buds, and it is of questionable use for vegetative buds. In an attempt to find an alternative test, we examined whether the dormancy state of vegetative and floral buds of trees exposed to different temperature conditions during the rest period is directly related to their ATP, ADP and NTP concentrations and ATP/ADP ratio. Once the buds had entered endo- or paradormancy, the nucleotide concentrations and the ATP/ADP ratio were low in the vegetative primordia and very low in the floral primordia. Only after the action of chilling, when the buds were considered to have completed the endodormancy and paradormancy phases, did the nucleotide concentrations increase, accompanied by a steep rise in ATP/ADP ratio. We conclude that the ATP/ADP ratio could be used to characterize the bud dormancy state by comparison with critical values of 1.5 for vegetative primordia and 1.0 for floral primordia.

Journal Article↗

PubMed1999

Candida albicans and Yarrowia lipolytica as alternative models for analysing budding patterns and germ tube formation in dimorphic fungi.

The site for bud selection and germ tube emission in two yeasts, Candida albicans and Yarrowia lipolytica, was analysed. Both dimorphic organisms display different patterns of budding, which also differ from those described for Saccharomyces cerevisiae. C. albicans, which is diploid and (until now) lacks a known sexual cycle, buds in an axial budding pattern. During the yeast-hypha transition induced by pH, serum, N-acetylglucosamine (GlcNAc) or temperature, germ tube emergence occurs at approximately 50% in a polar manner, while the other 50% of cells show non-polar germ tube emission. Y. lipolytica, in which most of the natural isolates are haploid and which has a well characterized sexual cycle, buds with a polar budding pattern independently of the degree of ploidy. Germ tube emission during the yeast-hypha transition in both haploid and diploid cells generally occurs at the pole distal from the division site (bipolar). The addition of hydroxyurea (HU), an inhibitor of DNA synthesis, also produces different effects. In its presence, and therefore in the absence of DNA synthesis, the yeast-hypha transition is completely abolished in Y. lipolytica. By contrast, in C. albicans germ tube emission in the presence of HU is similar to that observed in control cultures for at least 90 min under induction conditions. These results demonstrate that, rather than a single developmental model, several models of development should be invoked to account for the processes involved in the morphological switch in yeasts (the yeast-hypha transition).

Candida albicans↗

PubMed2003

Budding of melanized Cryptococcus neoformans in the presence or absence of L-dopa.

Cryptococcus neoformans is a pathogenic fungus that produces melanin when incubated in the presence of certain phenolic substrates such as L-3,4-dihydroxyphenylalanine (L-dopa). Melanin is an enigmatic polymer that is deposited in the cell wall and contributes to virulence. Substantial progress has been made in understanding the synthesis of melanin and the mechanisms by which it contributes to virulence, but relatively little is known about how melanin is rearranged during growth and budding. In this study we used transmission and scanning electron microscopy and immunofluorescence of melanized cells and melanin 'ghosts' to study the process of melanization during replication. Budding in melanized C. neoformans results in focal disruption of cell-wall melanin at the bud site. In the presence of L-dopa, bud-related melanin defects are repaired and daughter cells are melanized. However, in the absence of substrate, mother cells cannot repair their melanin defects and daughter cells are non-melanized. Hence, melanin in the parent cell is not carried to the daughter cells, but rather is synthesized de novo in buds. These results imply that melanin remodelling occurs during cell growth in a process that involves degradation and synthesis at sites of budding.

Cell Division↗

PubMed1999

Localization of Bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site.

Yeast cells of different cell type exhibit distinct budding patterns that reflect the organization of the actin cytoskeleton. Bud1p (Rsr1p), a Ras-like GTPase, and Bud2p, a GTPase-activating protein for Bud1p, are essential for proper budding pattern. We show that Bud2p is localized at the presumptive bud site in G(1) cells in all cell types and that this localization is independent of Bud1p. Bud2p subsequently localizes to the mother-bud neck after bud emergence; this localization depends on the integrity of the septins. These observations indicate that Bud2p becomes positioned in G(1) cells by recognizing cell type-specific landmarks at the presumptive bud site.

Cell Cycle Proteins↗

PubMed1969

Prunin identification, biological activity and quantitative change in comparison to naringenin in dormant peach buds.

Prunin (naringenin 7-glucoside), which was identified in dormant peach buds, was found to act as a growth inhibitor of wheat coleoptile elongation.Quantitative estimation on a fresh weight basis of the flavonoid in peach buds revealed a high level in late summer, a steep decrease in autumn that became more moderate during the winter, with lowest values near bud swell in spring. Two periods of reaccumulation of prunin during October and January fitted 2 periods of arrested growth of the resting bud. A negative correlation was demonstrated between prunin and naringenin levels during August through October. It is proposed that prunin might act as a precursor for naringenin that accumulates in resting buds in autumn. It is suggested that the system glucoside-aglucone-beta glucosidase might have regulatory properties in the dormant bud.

Journal Article↗

PubMed1976

Endogenous abscisic Acid in relation to bud growth in alternate bearing ;valencia' orange.

An investigation was conducted into the relation of ABA (cis-trans-abscisic acid) in the dormant buds of alternate bearing ;Valencia' orange (Citrus sinensis [L.] Osbeck) trees. ABA did not appear to be related to alternate bearing but t-ABA (2-transabscisic acid) did. There was 5- to 10-fold more t-ABA than ABA in the buds. There was more t-ABA in the buds of the "on" trees than in the buds of the "off" trees, and a drastic drop in t-ABA in both types of buds as spring growth approached. Bud dormancy and readiness for growth as related to t-ABA are discussed.

Journal Article↗

PubMed1984

Gibberellin A(4/7) and the Promotion of Flowering in Pinus radiata: Effects on Partitioning of Photoassimilate within the Bud during Primordia Diferentiation.

Gibberellin A(4/7) mixture (GA(4/7)), a highly effective promoter of early and enhanced flowering in the Pinaceae, caused a significant reallocation of dry matter and (14)C-photosynthate within terminal buds of Pinus radiata D. Don within 8 days of hormone treatment. Treatment with GA(4/7) to terminal shoots of vigorous, potentially flowering mature grafted propagules reduced the flow of photoassimilated (14)C and dry matter into the terminal bud as a whole, but significantly increased the dry matter and (14)C allocated within the bud to developing long-shoot primordia (potential seed-cone buds). This was accomplished at the expense of the structural tissues, the apical dome region, and the vegetative branch buds. Although GA(3) caused a similar reallocation of dry matter within the terminal bud, it was significantly less effective than GA(4/7) thus appears to have, in addition to any nutrient diversion abilities, a distinct morphogenic function in sexual differentiation.

Journal Article↗