Desiccation tolerance in the moss Polytrichum formosum: physiological and fine-structural changes during desiccation and recovery

BACKGROUND AND AIMS: This study explores basic physiological features and time relations of recovery of photosynthetic activity and CO(2) uptake following rehydration of a desiccation-tolerant moss in relation to the full temporal sequence of cytological changes associated with recovery to the normal hydrated state. It seeks reconciliation of the apparently conflicting published physiological and cytological evidence on recovery from desiccation in bryophytes. METHODS: Observations were made of water-stress responses and recovery using infrared gas analysis and modulated chlorophyll fluorescence, and of structural and ultrastructural changes by light and transmission electron microscopy. KEY RESULTS: Net CO(2) uptake fell to zero at approx. 40 % RWC, paralleling the fluorescence parameter PhiPSII at 200 micromol m(-2) s(-1) PPFD. On re-wetting the moss after 9-18 d desiccation, the initially negative net CO(2) uptake became positive 10-30 min after re-wetting, restoring a net carbon balance after approx. 0.3-1 h. The parameter F(v)/F(m) reached approx. 80 % of its pre-desiccation value within approx. 10 min of re-wetting. In the presence of the protein-synthesis inhibitors chloramphenicol and cycloheximide, recovery of F(v)/F(m) (and CO(2) exchange) proceeded normally in the dark, but declined rapidly in the light. Though initial recovery was rapid, both net CO(2) uptake and F(v)/F(m) required approx. 24 h to recover completely to pre-desiccation values. The fixation protocols produced neither swelling of tissues nor plasmolysis. Thylakoids, grana and mitochondrial cristae remained intact throughout the drying-re-wetting cycle, but there were striking changes in the form of the organelles, especially the chloroplasts, which had prominent lobes and lamellar extensions in the normally hydrated state, but rounded off when desiccated, returning slowly to their normal state within approx. 24 h of re-wetting. Sub-cellular events during desiccation and re-wetting were generally similar to those seen in published data from the pteridophyte Selaginella lepidophylla. CONCLUSIONS: Initial recovery of respiration and photosynthesis (as of protein synthesis) is very rapid, and independent of protein synthesis, suggesting physical reactivation of systems conserved intact through desiccation and rehydration, but full recovery takes approx. 24 h. This is consistent with the cytological evidence, which shows the thylakoids and cristae remaining intact through the whole course of dehydration and rehydration. Substantial and co-ordinated changes in other cell components, which must affect spatial relationships of organelles and metabolic systems, return to normal on a time span similar to full recovery of photosynthesis. Comparison of the present data with recently published results suggests a significant role for the cytoskeleton in desiccation responses.     

Asexual reproduction and protonemal development in vitro in Fontinalis antipyretica Hedw.

As sporophytes are very rare and asexual propagules unknown it is generally assumed that the moss Fontinalis antipyretica spreads almost exclusively via detached shoots carried in water currents. This study of regeneration in vitro reveals that F. antipyretica produces a variety of filament systems originating from virtually every part of the gametophyte, including: cortical cells in the bases of detached shoots, the margins and abaxial surfaces of leaves, stems following leaf removal and the laminae of detached leaves. The filaments vary from protonemata comprising short rectangular cells with transverse cross-walls to unbranched rhizoids. Filamentous gemmae, liberated by schizolysis, and spherical brood cells are produced in ageing and desiccating cultures. In nature these asexual propagules are probably produced in response to falling water levels and may have an important and hitherto unsuspected role in the spread and spatial genetic structure of F. antipyretica. In F. antipyretica the high level of phenotypic plasticity, that characterizes the mature gametophytes of aquatic bryophytes, also extends to the filamentous phase in the life cycle. Major differences between axenic and contaminated cultures are likely due to positive associations between the moss and bacterial and/or fungal contaminants.     

Tissue-specific,light-regulated and plastid-regulated expression of the single-copy nuclear gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana

The single-copy PetC gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana consists of five exons interrupted by four introns and encodes a protein of 229 amino acid residues with extensive sequence similarity to the chloroplast Rieske proteins of other higher plants. The N-terminal 50 amino acid residues constitute a presequence for targeting to the chloroplast and the remaining 179 amino acid residues make up the mature protein. Three of the introns are in identical positions in the PetC gene of Chlamydomonas reinhardtii, suggesting that they are of ancient origin. RNA-blot hybridisation showed that the gene was expressed in shoots, but not roots, and was light regulated and repressed by sucrose. The expression of chimeric genes consisting of PetC promoter fragments fused to the beta-glucuronidase (GUS) reporter gene was examined in A. thaliana and tobacco. In A. thaliana, GUS activity was detected in leaves, stems, flowers and siliques, but not in roots, and showed a strong correlation with the presence of chloroplasts. In transgenic tobacco, low levels of GUS activity were also detected in light-exposed roots. GUS activity in transgenic tobacco seedlings was light regulated and was decreased by norflurazon in the light suggesting regulation of PetC expression by plastid signals.     

Interactions of neural glycosaminoglycans and proteoglycans with protein ligands: assessment of selectivity, heterogeneity and the participation of core proteins in binding

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8- fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400- fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.      

Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales

Powerful analyses of population structure require information from multiple genetic loci. To help develop a molecular toolbox for obtaining this information, we have designed universal oligonucleotide primers that span conserved intron-exon junctions in a wide variety of animal phyla. We test the utility of exon-primed, intron-crossing amplifications by analyzing the variability of actin intron sequences from humpback, blue, and bowhead whales and comparing the results with mitochondrial DNA (mtDNA) haplotype data. Humpback actin introns fall into two major clades that exist in different frequencies in different oceanic populations. It is surprising that Hawaii and California populations, which are very distinct in mtDNAs, are similar in actin intron alleles. This discrepancy between mtDNA and nuclear DNA results may be due either to differences in genetic drift in mitochondrial and nuclear genes or to preferential movement of males, which do not transmit mtDNA to offspring, between separate breeding grounds. Opposing mtDNA and nuclear DNA results can help clarify otherwise hidden patterns of structure in natural populations.      

Defensive behaviour and its inheritance in the anabantoid fish, Macropodus opercularis and Macropodus opercularis concolor

In this preliminary study defense behaviour patterns (fear responses) are described in two closely related, behaviourally different inbred labyrinth fish subspecies and in their F₁ generation. The subspecies M. opercularis (characterized briefly by “active escape”) and M. opercularis concolor (characterized by “passive escape”) showed specific differences in the manifestation of certain defense behaviour patterns. In the F₁ hybrid generation dominance and overdominance of M. opercularis was found in most defense behaviour patterns. Analysing the frequencies and sequences of movement patterns it could be shown that defensive behaviour is not a random or entirely “plastic” process but that there is sequential linkage between the patterns and they form characteristic clusters. Our results suggest that manifestations of different patterns are under genetic control and presumably, genetic determination of certain patterns is not very complex. Attempts were made to determine whole brain noradrenaline, serotonine and dopamine levels of the two subspecies and a significant difference was found in the noradrenaline content.     

The Formation of Catenate Foliar Gemmae and the Origin of Oil Bodies in the Liverwort Odontoschisma denudatum (Mart.) Dum. (Jungermanniales): a Light and Electron Microscope Study

This article describes the ultrastructural events associatedwith the differentiation and liberation of the exogenous gemmaeproduced in branched acropetal chains along the margins of theleaves in the liverwort Odontoschisma denudatum. Formation ofa dorsal protrusion from young leaf cells containing a largecentral nucleus, small vacuoles, starch-free chloroplasts, scatteredcytoplasmic lipid droplets but no oil bodies, signals the onsetof the formation of the initial cell of a gemmiferous filament.The protrusion enlarges and the nucleus migrates into its base,therein dividing with the equator of the spindle virtually fillingthe central isthmus between the leaf surface and the now swollentip of the initial cell. Subsequent divisions of the initialcell produce a chain of cells in atropetal succession. Transverselyorientated microtubules line the cortical cytoplasm along thelateral walls of the terminal cells of the gemmiferous filaments,but are absent from the tips, thus suggesting that these cellselongate by intercalary, rather than by tip, growth. Duringmitosis microtubules are closely associated with the envelopesof spindle-shaped prophase nuclei, radiate from ill-definedspindle poles surrounded by plastids at metaphase and anaphaseand form a dense phragmoplast array during telophase. Pre-prophasebands are absent and it may be that the nuclear equator determinesthe plane of division in gemmiferous filaments. Chloroplastdivision, associated with extremely transient plastid-dividingrings, takes place during interphase. Lateral branches of thegrowing filaments arise from subapical cells by reiterationof the first division mechanism. Immediately following the proliferative divisions, which takeplace in cells measuring only 5-6 µm in diameter, oilbodies suddenly appear as flat pleomorphic cisternae associatedwith endoplasmic reticulum and occasional microtubules. Theircontents are electron-transparent apart from scattered osmiophilicdroplets. Throughout their ontogeny the oil bodies are closelyassociated with cytoplasmic lipid bodies but there is no evidenceof fusion. The nascent oil bodies swell rapidly to their finaldiameter, become ovoid to spherical in outline and are eventuallysuspended by fine cytoplasmic bridges within the vacuoles. Thelatter rapidly increase in size together with an expansion ofthe cells themselves until these reach their final diameterand length. The final event in gemma maturation is an endogenousdivision with the formation of a new internal wall along a phragmosome.Separation of the bicellular gemmae proceeds basipetally andinvolves the appearance of an electron-transparent line alongthe middle lamella in the cross walls, which often develop convexthickenings, and severing of the plasmodesmata. After theirliberation shallow scars are visible on the leaf surface underthe SEM. Gemma maturation sees a marked increase in the electron-opacityof the walls and dense staining of these together with Golgivesicles with the periodic acid/thiocarbohydrazide/silver proteinatetest for non-cellulosic carbohydrates. This change in wall chemistryand ultrastructure may be related to the fact that the maturinggemmae become extremely water repellant and are probably dispersedeither on the surface of water films or in the air.Copyright1995, 1999 Academic Press Bryophyta, gemmae, liverwort, microtubules, morphogenesis, oil bodies, polar growth, vegetative reproduction