Growth,cell wall formation and differentiation in the protonema of the moss,Funaria hygrometrica: Effects of plasmolysis on the developmental program and its expression

Summary Cultivation ofFunaria protonemata under plasmolytic or slightly subplasmolytic conditions initially causes a cessation of growth which is accompanied by a transient disappearance (or strong reduction in frequency, respectively) of putative cellulose synthesizing particle rosettes in the plasma membrane. Simultaneously, the formation and exocytosis of cell wall materialsecreting Golgi vesicles is slowed down. The latter process does not become apparent for several hours, though the reduction in activity can be proved indirectly. As a consequence of the imbalance between exocytosis, cell wall material accumulates in the plasmolytic space, generally at the cell tip. This indicates that the pattern of local, polar deposition of cell wall formation and cell elongation, membrane debris as well as wall material is maintained for some time. Later, however, the whole protoplast may become covered by new wall layers. Potentially growing filament tips and the distal region of nontip cells increase in diameter after longer cultivation in subplasmolytic conditions. It is suggested that normal wall growth results from a softening of the existing wall, its stretching and simultaneous stabilization by the apposition of new wall layers. We believe that the swelling is caused by a change in the equilibrium between the obviously less affected softening process and the imperfect stabilization by new wall layers because the wall layers which are formed at reduced turgor pressure are looser than normal and may have a changed composition.Kinetin-induced buds do not develop under plasmolytic conditions. Instead, spiral filaments are formed which readily give rise to buds when the osmotic value of the (kinetin-containing) medium is normalized. The results show that plasmolysis affects the expression of the developmental program rather than its initiation or maintenance.     

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.     

仙鹤藓属(Atrichum)藓类植物组织培养再生体系的建立

报道了仙鹤藓(Atrichum undulatum(Hedw.)P.Beauv.)和仙鹤藓小形变种(Atrichum undulatum var.minus(Hedw.)Par.)的组织培养再生体系的建立.为研究仙鹤藓属(Atrichum)藓类愈伤组织的诱导和再分化,将仙鹤鲜和仙鹤藓小形变种原丝体接种在含有4%葡萄糖和0.2～2.0 mg/L 6-BA的MS培养基上,培养一个月后,成功地诱导出疏松、易碎的绿色愈伤组织.愈伤组织诱导和常规继代培养较适合的培养基为含4%葡萄糖和1～2 mg/L6-BA的MS培养基.当将继代培养5次的脱分化藓类愈伤组织转移到含4%葡萄糖但无任何激素的MS培养基上时,能再分化形成原丝体,而在无任何碳源的Benecke培养基上培养时,能再分化形成经原丝体阶段发育来的直立配子体.     

不同培养基及酶对立碗藓原丝体的作用研究

采用含不同成分的MS培养基处理立碗藓的原丝体,发现随蔗糖浓度的提高原丝体的生长相对趋于老化;当培养基中附加不同浓度的2,4-D和6-BA 时,10日龄的原丝体出现了明显变化,当二者浓度均为0.5 mg*L-1,幼嫩的原丝体上能长出疏松易碎的白绿色愈伤组织.利用1%纤维素酶和0.8%的果胶酶分别处理10日龄、20日龄的原丝体和10日龄的茎叶体的叶片2～10 h,结果发现10日龄的原丝体在酶解2 h后能游离出大量的原生质体;而20日龄的原丝体效果较差,只能游离出极少量的原生质体;对于10日龄的茎叶体酶解10 h仍看不到原生质体游离出来.这表明立碗藓原丝体对不同的外界条件可产生不同的反应,但原丝体的生理状态不同对激素和酶类的反应具有非常显著的差别.     

Cloning and characterization of micro-RNAs from moss

Micro-RNAs (miRNAs) are one class of endogenous tiny RNAs that play important regulatory roles in plant development and responses to external stimuli. To date, miRNAs have been cloned from higher plants such as Arabidopsis, rice and pumpkin, and there is limited information on their identity in lower plants including Bryophytes. Bryophytes are among the oldest groups of land plants among the earth's flora, and are important for our understanding of the transition to life on land. To identify miRNAs that might have played a role early in land plant evolution, we constructed a library of small RNAs from the juvenile gametophyte (protonema) of the moss Physcomitrella patens. Sequence analysis revealed five higher plant miRNA homologues, including three members of the miR319 family, previously shown to be involved in the regulation of leaf morphogenesis, and miR156, which has been suggested to regulate several members of the SQUAMOSA PROMOTER BINDING-LIKE (SPL) family in Arabidopsis. We have cloned PpSBP3, a moss SPL homologue that contains an miR156 complementary site, and demonstrated that its mRNA is cleaved within that site suggesting that it is an miR156 target in moss. Six additional candidate moss miRNAs were identified and shown to be expressed in the gametophyte, some of which were developmentally regulated or upregulated by auxin. Our observations suggest that miRNAs play important regulatory roles in mosses.     

Dry/wet cycling reduces spore germination and viability in six peatland bryophytes

• Dry/wet cycling driven by water level fluctuation in wetlands may strongly influence the destiny of seeds. However, how dry/wet cycling affects spore survival and germinability in peatland bryophytes is poorly understood.
• Six peatland bryophytes, three hummock- and three hollow-dwelling Sphagnum species, were chosen as study species. We tested the effects of dry (60% air RH)/wet (waterlogging) cycle frequency (once per 12, 8 or 4 days for low, medium or high, respectively) and ratio (3:1, 1:1 or 1:3 dry:wet time per cycle) on spore germinability, viability, dormancy percentage and protonema development.
• Dry/wet cycling significantly reduced spore germination percentage and viability and slowed protonema development in all Sphagnum species, being more pronounced with higher dry/wet cycling frequencies. The hummock species S. capillifolium and S. fuscum had higher spore germination percentage after the continuous dry treatment, while the hollow species S. angustifolium, S. squarrosum and S. subsecundum showed the opposite response, compared to the continuously wet treatment. Except for S. squarrosum, spore viability was higher after the dry than after the wet treatment. Spore viability and dormancy percentage were higher after a dry/wet ratio of 1:3 than after ratios of 3:1 and 1:1.
• Our study shows that both germinability and viability of bryophyte spores are reduced by dry/wet cycling (especially when frequent) in peatlands. This emphasizes the need to ensure constant water levels and low frequencies of water level fluctuation, which are relevant in connection with wetland restoration, to promote Sphagnum spore survival and establishment in peatlands after disturbances.

     

Preprophase Band in Fern Protonemata: Mechanism of Development and Comparison with Flowering Plants

Adiantum capillus-veneris L. This review focuses on mechanisms of preprophase band development in fern protonemata. Received 12 December 1999/ Accepted in revised form 18 January 2000     

Photoinduction of circular F-actin on chloroplast in a fern protonemal cell

Summary Circular F-actin on a photooriented chloroplast was observed by rhodamine-phalloidin staining in the fernAdiantum protonemal cells in which phytochrome- or blue light receptor-mediated intracellular photoorientation of chloroplasts was induced. The circular structure located along the edge of chloroplast on the side facing the plasma membrane but not on the opposite side. Most of the chloroplasts in protonemal cell have dumbbell-shape and the circular ring-like structure was found on each half of the dumbbell. The structure was not observed in the cells which were kept in the dark, indicating the change of F-actin organization by the light condition. Possible role of the structure on the anchorage of chloroplast in its intracellular photoorientation was discussed.     

Photoorientation of chloroplasts in protonemal cells of the fernAdiantum as analyzed by use of a video-tracking system

Photoorientation of chloroplasts mediated by phytochrome and blue light-absorbing pigment in protonemal cells of the fernAdiantum was studied by use of inhibitors of the cytoskeleton and was analyzed with a video-tracking system. The photoorientation responses were inhibited by cytochalasin B and by N-ethylmaleimide (NEM) but not by colchicine, suggesting that the photomovement depends on the actomyosin system. In the dark, chloroplasts moved randomly, being independent of one another. After induction of photoorientation by polarized red light, most chloroplasts that had been located at the margin of cells moved almost perpendicularly to the cell axis toward the site of photoorientation. This type of movement was hardly ever observed in the dark. Under polarized blue light, such specific movements were less evident but were still observed in the case of a few chloroplasts. After photoorientation was complete, chloroplasts still moved in random directions but their mobility was lower than that in the dark, indicating the presence of some anchoring mechanism. When EGTA was applied, photoorientation was inhibited but this inhibition was overcome by the addition of CaCl₂. Video-tracking of chloroplasts in the dark revealed that the mobility of chloroplasts was higher in medium with EGTA than in medium with EGTA plus CaCl₂ and that many of the chloroplasts moved jerkily in the medium with EGTA. This change in the nature of movements was also seen under polarized light, resulting in the disturbance of photoorientation. These results indicate that the inhibition of photoorientation at low concentrations of Ca²⁺ ions may be due to change in the nature of chloroplast movement.