On the action mechanism of cytokinins in mosses: Caulonema specific proteins

Soluble proteins extracted with Tris-buffer pH 8.8 from both differentiation stages of the protonema of Funaria hygrometrica (chloronema and caulonema) were separated by microgel electrophoresis. 4x10^-3 mg protein/gel was applied. The caulonema, which is the only part of the protonema able to form buds following cytokinin treatment, contained 3 protein bands, which were absent in chloronema. We designate them as caulonema specific proteins (CSP, approximate molecular weight 500,000). The CSP disappear when the caulonema is isolated and its cells regenerate to chloronema. The CSP bind kinetin (6 Furfurylamino [8-¹⁴C]purine) or BAP (6-benzylamino[8-¹⁴C]purine) about 10 times stronger than the remaining protein bands in the gel. The greatest part of the cytokinin is metabolized in a short time and consequently a part of the activity in the gel is incorporated into RNA and removable with RNase. Simultaneous application of adenosine and cytokinin reduced the incorporation of radioactivity into RNA and enhanced the specific activity in one of the CSP bands. In all other bands it remained unchanged.From the results it can be suggested that the CSP are probably involved in the early reactions to cytokinin of the target cells.Abbreviations CSP Caulonema specific proteins - BAP 6-benzylaminopurine - GA₁ gibberellin A₁     

Cytokinin induces the developmentally restricted synthesis of an extracellular protein in Physcomitrella patens

Early development of the moss Physcomitrella patens follows a simple course leading to the formation of a filamentous protonema containing only two cell-types, chloronema and caulonema. The addition of the hormone cytokinin leads to the induction of multicellular buds from such protonema. The spectrum of extracellular proteins (ECPs) synthesized by P. patens has been investigated at defined stages of development and under defined hormone treatments. It is found that in contrast to the limited changes in intracellular protein synthesis detectable, in the extracellular environment major and specific changes in the patterns of proteins synthesized occur. For example, the presence of caulonema cells is characterized by the synthesis of a 25 kDa ECP whereas early chloronema differentiation is distinguished by the presence of a 38 kDa ECP. The analysis of the pattern of ECPs synthesized by developmental mutants altered in bud formation, and in response to cytokinin in tunicamycin treated protonema (in which bud induction is blocked) indicate that the synthesis of a 14 kDa ECP is specifically induced by cytokinin. This protein represents a novel cytokinin-induced ECP. These data show that the differentiation of particular cell types in plants is associated with the synthesis of particular ECPs, and suggest that hormones which induce specific morphogenic events may do so via the synthesis of specific ECPs.     

Occurrence and biosynthesis of auxin in protonema of the moss Funaria hygrometrica

We have investigated the presence of auxin and the ability of chloronema cells to synthesize indole-3-acetic acid (IAA) in axenic protonema cell cultures of the moss Funaria hygrometrica. The endogenous level of auxin activity was 4 and 7μg-IAA equivalents/kg in caulonema and chloronema cell types, respectively. Based on an indole-α-pyrone fluorometric assay, the level of putative IAA was observed to be 5.0 and 1.9.μg/kg in caulonema and chloronema cells, respectively. [³H]Tryptophan was metabolized into IAA via the indole-pyruvate pathway by intact chloronema cells and also by the cell free homogenates. More [³H]IAA accumulated when homogenates from cells pre-grown at low cell densities (< 0.5 mg/ml) as compared to those at high cell densities ( > 0.5 mg/ml) were used. Since the activities of peroxidase and IAA-oxidase are known to be high at high cell densities, the lack of accumulation of radioactivity in IAA at high densities can be attributed to a high level of IAA-oxidizing enzymes. Our results suggest a possible relationship between IAA accumulation and caulonema differentiation.     

Characteristics of spore germination and protonemal development in Hypnum pacleseens

The spore germination, protonemal development, and gametophyte differentiation of Hypnum pacleseens were observed in cultivation. Photomicrographs showed that spore germination of Hypnum pacleseens occured within the exospore. Its protonema is massive with filamentous chloronema formed inside. The terminal part of the chloronema differentiated into filamentous caulonema and its rhizoid was derived from the apical cell of the filamentous chloronema. The initial cell of gametophyte differentiated from chloronema and caulonema. Sporeling-type of Hypnum pacleseens is developmentally similar to Glyphmitrium-type.     

Photoaffinity labelling with the cytokinin agonist azido-CPPU of a 34 kDa peptide of the intracellular pathogenesis-related protein family in the moss Physcomitrella patens

As in higher plants, the development of the moss Physcomitrella patens is regulated by environmental signals and phytohormones. At the protonema level transition from chloronema to caulonema cells is under auxin control. The formation on second sub-apical caulonema cells of buds that will give rise to the leafy gametophore requires cytokinins. Using [³H]azidoCPPU (1-(2-azido-6-chloropyrid-4-yl)-3-(4-[³H])phenylurea), a photoactivatable cytokinin agonist, we have specifically photolabelled a soluble 34 kDa protein of P. patens. Urea derivatives were very efficient competitors of photolabelling while purine-type cytokinins were poor competitors. The protein UBP34 was purified by affinity chromatography and the sequences of six internal peptides obtained. A cDNA encoding UBP34 was cloned by screening a P. patens protonema cDNA library with a probe amplified by PCR using degenerate primers designed from the peptide sequences. The UBP34 amino acid sequence shows an average sequence identity of 42% with both intracellular PR proteins and the BetV1-related family of plant allergens. Recombinant UBP34 expressed in Escherichia coli was confirmed to bind azidoCPPU.     

Charakterisierung des Regenerationsprozesses im Caulonema eines Mooses durch autoradiographische Bestimmung der DNS-Synthese

Summary Ten-cell-long filaments of the caulonema of Funaria hygrometrica were isolated and labeled with ³H-thymidine. During the process of regeneration this precursor is incorporated into the nucleus and the chloroplasts. The nuclei of aged cells are preferentially labeled, even the nuclei of such cells which probably will no longer divide.From these facts it is concluded that DNA synthesis can occur during the process of regeneration irrespectively of a following cell division.     

碎米藓属两种藓类植物孢子萌发与原丝体发育特征研究

为获取其孢子萌发类型与该属植物系统发育、生态选择以及生殖策略选择的相关性,该研究通过室内人工培养的方式,在微米量级下观察并描述了碎米藓属(Fabronia)碎米藓(F.pusilla)和东亚碎米藓(F.matsumurae)两种藓类植物孢子萌发、原丝体发育和配子体发生的过程.结果表明:(1)两种藓类植物孢子均为壁外萌发...     

Induction of budding on chloronemata and caulonemata of the moss,Physcomitrella patens,using isopentenyladenine

The bud-inducing effect of the cytokinin N⁶-(²-isopentenyl)-adenine (i⁶-Ade) was examined in the moss Physcomitrella patens growing in liquid culture. Under these conditions, buds could be induced on chloronemata as well as on caulonemata. By application of i⁶-Ade, bud-formation was accelerated in both types of tissue. The number of buds, their size and their site of development were dependent on the concentration of the cytokinin in the range of 10^-7 M to 10^-5 M. Moreover, the percentage of caulonema cells increased with a cytokinin concentration of 10^-5 M. These results indicate that chloronema cells may also function as target cells for exogenous cytokinins. The composition of proteins from caulonemata and chloronemata of two different species (P. patens and Funaria hygrometrica), grown on solid medium were compared. No differences could be detected between the protein patterns of caulonemata and chloronemata of the same species while between the two species the differences were obvious.Abbreviations i⁶-Ade N⁶-(²-isopentenyladenine) - Da dalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The occurrence of caulonema-specific proteins in different moss species

Soluble proteins extracted with Tris-buffer pH 8.8 from different mosses are analysed by microgelelectrophoretic method for comparison to the specific proteins present only in the caulonema of Funaria hygrometrica. The protein patterns are also compared with those of liverwort, fern gametophytes and sporophytes and tobacco. It is observed that the caulonema specific proteins are only present in the caulonema of these mosses and are absent in other plants.Abbreviations CSP caulonema specific proteins     

Unique Tissue-Specific Cell Cycle in Physcomitrella

Abstract: The moss Physcomitrella patens (Hedw.) B.S.G. is a novel tool in plant functional genomics as it has an inimitable high gene targeting efficiency facilitating the establishment of gene/function relationships.
Here we report, based on flow cytrometric (FCM) data, that the basic nuclear DNA content per cell of Physcomitrella is 0.53 pg, equating to a genome size of 1 C = 511 Mbp. Furthermore, we describe a unique tissue-specific cell cycle change in this plant. Young plants consisting of only one cell type (chloronema) displayed one single peak of fluorescence in FCM analyses. As soon as the second cell type (caulonema) developed from chloronema, a second peak of fluorescence at half the intensity of the previous one became detectable, indicating that caulonema cells were predominantly at the G1/S transition, whereas chloronema cells were mainly accumulating at the G2/M transition. This conclusion was validated by further evidence: i) The addition of ammonium tartrate arrested Physcomitrella in the chloronema state and in G2/M. ii) Two different developmental mutants, known to be arrested in the chloronema/caulonema transition, remained in G2/M, regardless of age and treatment. iii) The addition of auxin or cytokinin induced the formation of caulonema, as well as decreasing the amount of cells in G2/M phase. Additionally, plant growth regulators promoted endopolyploidisation.
Thus, cell cycle and cell differentiation are closely linked in Physcomitrella and effects of plant hormones and environmental factors on both processes can be analysed in a straight forward way. We speculate that this unique tissue-specific cell cycle arrest may be the reason for the uniquely high rate of homologous recombination found in the Physcomitrella nuclear DNA.     

Characteristics of spore germination and protonemal development in Hypnum pacleseens

The spore germination,protonemal development,and gametophyte differentiation of Hypnum pacleseens were observed in cultivation.Photomicrographs showed that spore germination of Hypnum pacleseens occured within the exospore.Its protonema is massive with filamentous chloronema formed inside.The terminal part of the chloronema differentiated into filamentous caulonema and its rhizoid was derived from the apical cell of the filamentous chloronema.The initial cell of gametophyte differentiated from chloronema and caulonema.Sporeling type of Hypnum pacleseens is developmentally similar to Glyphmitrium-type.     

尖叶拟船叶藓原丝体发育特征研究

将尖叶拟船叶藓[Dolichomitriopsis diversiformis(Mitt.)Nog.]孢子接种于Knop培养基上,置于恒温培养箱中培养,在光学显微镜下对其原丝体(protonema)发育特征进行了详细观察和记录。结果表明:孢子第2天就开始萌发,第6天时其萌发率达90%以上;原丝体系统由绿丝体(chloronema)和轴丝体(caulonema)构成,假根(rhizoides)产生于芽体基部,由轴丝体退化而成;配子枝原始细胞产生于绿丝体分枝的基部或轴丝体上的斜壁细胞;配子枝(game tophore)形成后其上各部位都可形成假根;孢子萌发类型为真藓型(Bryum-type)。     

On the formation of protonema ofDrummondia sinensis of the orthotrichaceae

In the present study, the mode of formation of the protonema ofDrummondia sinensis is recorded and the relationship between the sporeling type as a response to ecological adaptation is discussed. A spore germinates inside the stretched exospore, and later forms a massive protonema consisting of 8–20 cells. Soon after, the stretched spore coat ruptures and a mass of 5–6 cells protrdes from the broken exospore. In this species neither chloronema nor caulonema could be observed, although such is often seen in other species of Bryales. An initial cell of the leafy shoot is formed from the apical cell of the massive protonema.     

Distribution of activated calmodulin in the chloronema tip cells of the moss Funaria hygrometrica

Auxin (indole-3-acetic acid) regulates caulonema differentiation as a result of gradual transitional events in the chloronema tip cells in moss protonema. This auxin action in the moss Funaria hygrometrica involves a rapid influx of calcium ions from the extracellular medium. This investigation demonstrates spatial and temporal changes in calmodulin (CaM) activation (formation of Ca(2+)-CaM complex) in the chloronema tip cells subjected to auxin treatment. Photomicroscopic localisation of the fluorescence (excitation at 365 nm and emission of 397 nm) from the tricomplex of Ca(2+)-CaM with trifluoperazine (TFP, a blocker of Ca(2+)-CaM action) shows a tip to base (tip high) gradient of Ca(2+)-CaM in the chloronema tip cells. Comparison of Ca(2+)-CaM-TFP fluorescence over time in the chloronema tip cells of wild type Funaria with the response in an auxin overproducer mutant (86.1) and an auxin deficient mutant (87.13) reveals the involvement of auxin in calmodulin activation as a rapid response prior to cell differentiation.     

Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens

Tip growth is a mode of cell expansion in which all growth is restricted to a small area that forms a tip in an elongating cell. In green plants, tip growth has been shown to occur in root hairs, pollen tubes, rhizoids, and caulonema. Each of these cell types has a longitudinally elongated shape, longitudinally oriented microtubules and actin microfilaments, and a characteristic cytoplasmic organization at the growing tip which is required for growth. Chloronema are elongated cylindrical shaped cells that form during the development of the moss protonema. Since there are no published reports on the precise mode of chloronema elongation and conflicting interpretations of its cytology, the mechanism of cell growth has remained unclear. To determine if chloronema elongate by tip or diffuse growth, time-lapse light microscopy was employed to follow the movement of fluorescent microspheres attached to the surface of growing cells. It is shown here that chloronemal cells elongate by a form of tip growth. However, the slower growth of chloronema compared with caulonema is probably the result of differences in cytological organization of the growing tip.     

Polarity and growth of caulonema tip cells of the moss Funaria hygrometrica

In the caulonema tip cells of Funaria hygrometrica, chloroplasts, mitochondria, and dictyosomes have differences in structure which are determined by cell polarity. In contrast to the slowly growing chloronema tip cells the apical cell of the caulonema contains a tip body. Colchicine stops tip growth; it causes the formation of subapical cell protrusions, redistribution of the plastids, and a loss of their polar differentiation. Cytochalasin B inhibits growth and affects the position of cell organelles. After treatment with ionophore A23 187, growth is slower and shorter and wider cells are formed. D₂O causes a transient reversion of organelle distribution but premitotic nuclei are not dislocated. In some tip cells the reversion of polarity persists; they continue to grow with a new tip at their base. During centrifugation, colchicine has only a slight influence on the stability of organelle anchorage. The former polar organization of most cells is restored within a few hours after centrifugation, and the cells resume normal growth. In premitotic cells the nucleus and other organelles cannot be retransported, they often continue to grow with reversed polarity. Colchicine retards the redistribution of organelles generally and increases the number of cells that form a basal outgrowth. The interrelationship between the peripheral cytoplasm and the nucleus and the role of microtubules in maintaining and reestablishing cell polarity are discussed.Abbreviations DMSO dimethylsulfoxide - CB cytochalasin B Dedicated to Prof. Dr. A. Pirson on the occasion of his 70. birthday     

Uptake, transport and metabolism of cytokinin in moss protonema

Uptake, transport and metabolism of cytokinin in the protonemaof Funaria hygrometrica were studied using labelled kinetin(6-furfurylamino [8-¹⁴C]-purine). All cells of the protonema,chloronema and caulonema, were able to take up kinetin, whichwas carried in the symplastic transport system from cell tocell. Radioactivity was especially accumulated in growing cellsof the protonema. Kinetin was metabolized immediately afteruptake. While only very little kinetin (less than 1%) remainedas free kinetin and one part was immobilized in chromatographicseparation [e.g. attached to proteins and incorporated intonucleic acids (17)], most of the remaining kinetin was metabolizedto adenine derivatives. Exogenously supplied adenosine changedthe metabolism of kinetin. In the caulonema, adenosine reducedthe turnover of kinetin to other adenine derivatives and enhancedthe content of labelling in the start fraction. Thus adenosinecan stimulate cytokinin-dependent bud formation in moss protonema. (Received November 24, 1977; )     

Germination of the Spores and Development of Primary and Secondary Protonema of Funaria hygrometrica

Abstract

1. The primary protonema of Funaria hygrometrica, cultivated on Knop's or Marchal's agar in the light, proved to consist of filaments with much chlorophyll, a hyaline membrane, perpendicular cross-walls and branches equal to the main filament (chloronema). These filaments grow on the surface of the agar, the branches may also grow vertically. Sometimes filaments with less chlorophyll occur immediately after the germination. The caulonema described by Sironval has not been observed. Thus the rhizoid-like forms mentioned in the literature should more likely be considered as a result of external conditions (see Schoene, Bauer, Heitz and Fitting). Therefore it remains doubtful if a distinction between rhizoids and chloronema on the primary protonema is of any importance as it is impossible to give a good definition of either form.

At the base of moss plants main filaments with brown membranes, oblique septa and without chlorophyll may develop (rhizoids). They grow on the surface or within the agar. In F. hygrometrica especially, the stem seems to influence the occurrence of these rhizoids. The main filaments form buds on the basal cell of the branches and thus serve for vegetative reproduction. The branches show the characteristics of the chloronema. This is contrary to the conclusion of Westerdijk that rhizoids would pass into chloronema only when they are damaged or when the growth of the end bud of the plant is inhibited. At the base of the plant, moreover, little ramified, short branches with oblique septa appear which do not produce buds.

2. Branches may develop in the first growth stages of the primary protonema at any point of the cells. One single cell of a main filament can produce none, one, or more than one branch. Later the branches appear immediately behind the acroscopic cross-wall except in a few cases. Each cell then produces one branch.

3. Buds always develop at the basal cell of a primary branch of a green main filament or of a rhizoid derived from a moss plant.

4. In two ways the protonema may fall into pieces, which can develop into new main filaments:

(a) By forming brood cells; rounded cells which get detached by splitting of the septum. This phenomenon is very frequent. Contrary to Servettaz's opinion it seems to occur particularly under unfavourable conditions.

(b) By forming special cells, tmemata, whose walls are rent. These occur on the primary protonema contrary to the observations of Correns and Bauer, but they are much less frequent than the brood cells. No observations have been made on the circumstances of their occurrence.     

One evolutionarily selected amino acid variation is sufficient to provide functional specificity in the cold shock protein paralogs of Staphylococcus aureus

Bacterial genomes encode several families of protein paralogs. Discrimination between functional divergence and redundancy among paralogs is challenging due to their sequence conservation. Here, we investigated whether the amino acid differences present in the cold shock protein (CSP) paralogs of Staphylococcus aureus were responsible for functional specificity. Since deletion of cspA reduces the synthesis of staphyloxanthin (STX), we used it as an in vivo reporter of CSP functionality. Complementation of a ΔcspA strain with the different S. aureus CSP variants showed that only CspA could specifically restore STX production by controlling the activity of the stress-associated sigma B factor (σ^B). To determine the amino acid residues responsible for CspA specificity, we created several chimeric CSPs that interchanged the amino acid differences between CspA and CspC, which shared the highest identity. We demonstrated that CspA Pro58 was responsible for the specific control of σ^B activity and its associated phenotypes. Interestingly, CspC gained the biological function of CspA when the E58P substitution was introduced. This study highlights how just one evolutionarily selected amino acid change may be sufficient to modify the specific functionality of CSP paralogs.