Intact Fronds Research Articles

First Report of Agrobacterium rhizogenes-induced Hairy Root Formation in Selaginella bryopteris: a Pteridophyte Recalcitrant to Genetic Transformation

Abstract we report A. rhizogenes-induced hairy root formation in S. bryopteris, a medicinally and commercially important plant. A. rhizogenes strain LBA1334 co-cultivated with explants (root, rhizophore, stem portion near the root, and stem with intact fronds) for 24 and 48 h after transformation for induction of hairy roots. The induction of hairy root was observed after 6 days of infection in case of 48 h co-cultivation only. PCR with rolA and virC gene specific primers confirmed the induced hairy roots were due to Ri T-DNA integration and not due to contaminating A. rhizogenes. The root network as explants showed the maximum transformation efficiency. We tested different media like MS, SHFR (Stage Hog Fern Root) and KNOP’s during transformation for hairy root induction. The SHFR based media showed good response in transformation as well as propagation. Further, transformation efficiency was enhanced by addition of TDZ (2 mg/L) and Bevistin (0.1%) in SHFR media. The present work would be helpful in hairy roots-based in vitro production of secondary metabolites and on aspect of functional genomics of S. bryopteris.

Disturbance alters ecosystem engineering by a canopy-forming alga

Canopy-forming fucoid algae have an important role as ecosystem engineers on rocky intertidal shores, where they increase the abundance of species otherwise limited by exposure during low tide. The facilitative relationship betweenAscophyllum nodosumand associated organisms was explored using a frond breakage experiment (100%, 50%, 25%, 0% intact-frond treatments) in southern England, to assess the consequences of disturbance. Understorey substratum temperature was on average 3°C higher in 0% and 25% intact-frond treatments than in plots with 50% and 100% intact fronds. Light (as PAR during low tide) doubled in 0% intact-frond treatments in comparison to other treatments (which had similar light levels). Mobile invertebrate species richness declined by on average 1 species per m2in the treatments with only 25% and 0% intact fronds, and the abundance ofLittorina obtusatadeclined by 2.4–4.2 individuals per m2in the treatments with 25 and 0% intact fronds. Sessile taxa, includingOsmundea pinnatifidaand encrusting coralline algae, declined by half on average in the 0% intact-frond treatment. These results suggest that the ability ofAscophyllumto mediate environmental conditions to the understorey is the mechanism responsible for species distributed in the understorey (autogenic ecosystem engineering). The results of this study imply that a pulse disturbance resulting in a 50% breakage ofAscophyllumfronds significantly increases temperature and decreases the abundance of mobile invertebrates usually associated withAscophyllum. Sessile taxa associated withAscophyllumcan, however, withstand disturbances down to 25% intactAscophyllumfronds.

The role of peltate scales in desiccation tolerance of Pleopeltis polypodioides.

The extreme drought tolerance of the resurrection fern is in part the result of the dorsal scales that assist in water distribution and controlled desiccation. We studied the effect of peltate scales on water uptake and loss of the desiccation-tolerant epiphytic fern Pleopeltis polypodioides using optical and FTIR microscopy and staining with calcofluor, solophenyl flavine7GFE, and Ruthenium Red. We provide information on structure, property, and function of the scales by measuring water uptake and dehydration, contact angles, and metabolic activity. Peltate scales mainly contain cellulose, xylogalactans, and pectin. Water is absorbed from the center of scales, and the overlapping arrangement of scales facilitates surface spreading of water. Intact fronds hydrated fully within 5h of imbibition of the apical pinna, without scales water uptake stopped after 1h. Hydration rates via rhizomes followed a longer time course but also improved in the presence of scales. Fronds with and without scales lost half of their water content in 15 or 4h, respectively. The overall metabolism of rapidly dehydrated fronds was significantly reduced compared with slowly dehydrated fronds. Thus, water management and metabolism of Pleopeltis are dependent on surface properties determined by peltate scales.

Chemical settlement inhibition versus post-settlement mortality as an explanation for differential fouling of two congeneric seaweeds.

It has been proposed that seaweed secondary metabolites, e.g. brown algal phlorotannins, may have an ecologically important function as a chemical defence against epiphytes, by acting against colonisation of epiphytic organisms. We tested whether the low epiphytic abundance on the invasive brown seaweed Fucus evanescens, compared to the congeneric F. vesiculosus, is due to a more effective chemical defence against epiphyte colonisation. A field survey of the distribution of the common fouling organism Balanus improvisus (Cirripedia) showed that the abundance was consistently lower on F. evanescens than on F. vesiculosus. However, contrary to expectations, results from experimental studies indicated that F. vesiculosus has a more effective anti-settlement defence than F. evanescens. In settlement experiments with intact fronds of the two Fucus species, both species deterred settlement by barnacle larvae, but settlement was lower on F. vesiculosus both in choice and no-choice experiments. Phlorotannins from F. vesiculosus also had a stronger negative effect on larval settlement and were active at a lower concentration than those from F. evanescens. The results show that Fucus phlorotannins have the potential to inhibit settlement of invertebrate larvae, but that settlement inhibition cannot explain the lower abundance of the barnacle Balanus improvisus on F. evanescens compared to F. vesiculosus. Assessment of barnacle survival in the laboratory and in the field showed that this pattern could instead be attributed to a higher mortality of newly settled barnacles. Observation suggests that the increased mortality was due to detachment of young barnacles from the seaweed surface. This shows that the antifouling mechanism of F. evanescens acts on post-settlement stages of B. improvisus.

Morphological plasticity and apparent loss of apical dominance following the natural loss of the main apex in Pterocladiella capillacea (Rhodophyta, Gelidiales) fronds

In an intertidal population of Pterocladiella capillacea from southern Baja California, Mexico, fronds that lacked their main apex were bushier than intact fronds. A multivariate statistical analysis showed that the absence of the main apex was associated with higher frond biomass, a higher degree of branching, higher frond width, and longer main branches. The variation in frond form when the main apex is absent might be a response to the loss of apical dominance. The potential relevance of this morphological plasticity for the survival of thalli in the intertidal zone is discussed.

Production of bromoform and dibromomethane by Giant Kelp: Factors affecting release and comparison to anthropogenic bromine sources

Macrocystis pyrifera (Giant Kelp), a dominant macroalgal species in southern California, produced 171 ng per g fresh wt (gfwt) per day of CHBr3 and 48 ng gfwt−1 d−1 of CH2Br2 during laboratory incubations of whole blades. Comparable rates were measured during in situ incubations of intact fronds. Release of CHBr3 and CH2Br2 by M. pyrifera was affected by light and algal photosynthetic activity, suggesting that environmental factors influencing kelp physiology can affect halomethane release to the atmosphere. Data from H2O2 additions suggest that brominated methane production during darkness is limited by bromide oxidant supply. A bromine budget constructed for a region of southern California indicated that bromine emitted from the use of CH3Br as a fumigant (1 × 108 g Br yr−1) dominates macroalgal sources (3 × 106 g Br yr−1). Global projections, however, suggest that combined emissions of marine algae (including microalgae) contribute substantial amounts of bromine to the global cycle, perhaps on the same order of magnitude as anthropogenic sources.

Effects of harvesting onAscophyllum nodosum (L.) Le Jol. (Fucales, Phaeophyta): a demographic approach

Although populations ofAscophyllum nodosum are harvested commercially, little is known about the effects on demographic vital rates (growth, reproduction, survival). This study examines the effects of harvesting season and harvesting intensity on growth, reproduction and mortality of intact fronds in four size classes and in fronds truncated by the harvest. Knowledge of size-specific vital rates was used to evaluate the response of the population to harvesting.

Tetcyclacis and Abscisic Acid-Sensitive Reduction of Extracellular Ferricyanide by Mesophyll Cells ofValerianella locustaandLemna gibba

Electron transport across the plasma membrane of Valerianella locusta mesophyll cells and intact fronds of Lemna gibba, induced by 10 -3 M ferricyanide, was inhibited by tetcyclacis, an inhibitor regarded to be specific for cytochrome P-450 mono-oxygenases. The effect was dependent on the concentration of tetcyclacis and the duration of preincubation. The apparent rate of transmembrane electron transport increased in the presence of catalase, indicating tetcyclacis-induced H 2 O 2 -production or additional tetcyclacis-independent H 2 O 2 release

Degradation of the 32 kD Herbicide Binding Protein in Far Red Light

White light (400-700 nanometers) supports the activity of photosystem I (PSI) and photosystem II while far red light (>/=700 nanometers) supports PSI almost exclusively. In intact fronds of Spirodela oligorrhiza, turnover of the 32 kilodaltons herbicide binding protein is stimulated under both these light conditions, although not in the dark or at wavelengths >730 nanometers. As is the case in white light, the far red light induced degradation of the protein is inhibited by DCMU. The means by which far red light operates is unclear. Hypotheses considered include: PSI activated proteolysis, PSI-induced formation of semiquinone anions, and PSI-generated free radicals.

Effect of Light Quality on the Composition, Function, and Structure of Photosynthetic Thylakoid Membranes of Asplenium australasicum (Sm.) Hook

The effect of light quality on the composition, function and structure of the thylakoid membranes, as well as on the photosynthetic rates of intact fronds from Asplenium australasicum, a shade plant, grown in blue, white, or red light of equal intensity (50 microeinsteins per square meter per second) was investigated. When compared with those isolated from plants grown in white and blue light, thylakoids from plants grown in red light have higher chlorophyll a/chlorophyll b ratios and lower amounts of light-harvesting chlorophyll a/b-protein complexes than those grown in blue light. On a chlorophyll basis, there were higher levels of PSII reaction centers, cytochrome f and coupling factor activity in thylakoids from red light-grown ferns, but lower levels of PSI reaction centers and plastoquinone. The red light-grown ferns had a higher PSII/PSI reaction center ratio of 4.1 compared to 2.1 in blue light-grown ferns, and a larger apparent PSI unit size and a lower PSII unit size. The CO(2) assimilation rates in fronds from red light-grown ferns were lower on a unit area or fresh weight basis, but higher on a chlorophyll basis, reflecting the higher levels of electron carriers and electron transport in the thylakoids.The structure of thylakoids isolated from plants grown under the three light treatments was similar, with no significant differences in the number of thylakoids per granal stack or the ratio of appressed membrane length/nonappressed membrane length. The large freeze-fracture particles had the same size in the red-, blue-, and white-grown ferns, but there were some differences in their density. Light quality is an important factor in the regulation of the composition and function of thylakoid membranes, but the effects depend upon the plant species.

Long Distance Transport in Macrocystis integrifolia

Movement of THO and tritium-labeled photoassimilate was studied in intact fronds and frond cuttings of Macrocystis integrifolia following labeling of a mature blade by tritiated water. Both THO and tritium-labeled assimilate moved from the source blade to sink areas at velocities comparable to those recorded earlier for (14)C- and (32)P-labeled compounds. In intact fronds and frond cuttings, THO and tritium-labeled assimilate showed a declining gradient with increasing distance from the source. In the exudate collected from the basal cut end of the frond, there was a marked increase in radioactivity with time in the photoassimilate, but no such gradient was evident for THO. These results support the idea that, although both tritium-labeled assimilate and THO move in the sieve elements, THO is rapidly exchanged with water in the tissues surrounding the sieve elements. Finally, it is shown that THO is transported to the sink and there "unloaded"; indeed, it can move out of the plant itself. The data on velocity and directionality of transport as well as unloading of THO at the sink are discussed, along with computations on specific mass transfer, and favor the idea that Münch's pressure-flow hypothesis is applicable in Macrocystis for long distance translocation of photoassimilates.

Long Distance Transport in Macrocystis integrifolia

Long distance transport of (14)C-labeled photoassimilate was studied in Macrocystis integrifolia Bory. Movement of label followed the source-sink relationship; mature blades closer to the holdfast with young 2 degrees and 3 degrees fronds transported mostly to the base, those closer to the frond apex transported mostly to the apex, and those in intermediate positions transported both acropetally and basipetally. The velocity of movement of (14)C as computed both from study of intact fronds and exudate was in the range of 35 to 72 centimeters per hour and these estimates are on the low side. The composition of the translocate as determined from intact fronds was the same as that determined from exudate analysis; furthermore, this composition was nearly identical with that of the photosynthate (40 to 50% mannitol and 40 to 50% amino acids). From these data we conclude that the exudate represents the sieve tube sap and that there is little if any selectivity exercised in the loading and translocation of photoassimilate. An analysis of translocated label in the growing apex is presented and indicates that the synthesis of polymeric compounds such as laminaran, alginate, cellulose, lipids, and "protein" occurs in situ from the transported mannitol and amino acids. Detailed data on chemical composition of sieve tube sap from M. integrifolia and M. pyrifera (L.) C.A. Agardh are given and compared with the sieve tube sap from higher plants. Finally, we show that stipe segments, 60 to 100 centimeters long with three to six attached blades, are useful for translocation studies in Macrocystis.

Long Distance Transport in Macrocystis integrifolia

Discs from mature regions of Macrocystis blades picked up significantly more [(32)P]phosphate from the ambient medium than similar discs from young meristematic regions, and this uptake was higher in light than in darkness. Double-labeling experiments with NaH(14)CO(3) and [(32)P]phosphate, using intact fronds as well as cut frond segments, indicated that (32)P was translocated from mature blades to sink regions at velocities of 25 to 45 centimeters per hour, velocities comparable to (14)C translocation velocity in the same material. There was a slight delay in transport of (32)P which may be due to a delay in loading or to a high metabolism of (32)P in the transporting channels. Histoautoradiography of stipe segments in the translocation pathway indicated that transport of label occurred in the peripheral parts of medulla. An analysis of (32)P-labeled compounds in the fed blade and in the sieve tube sap, collected from basal cut ends of stipes, indicated major differences in labeling patterns. In the blade, a high proportion of (32)P was recovered as inorganic phosphate and relatively small amounts were found in hexose mono- and diphosphates, UDPG and ATP. In the sieve tube sap, however, only a small amount of (32)P was present as inorganic phosphate, a large proportion was found in hexose mono- and diphosphates, and appreciable amounts were present in ATP and UDPG.

KINETICS OF THE UPTAKE OF SOME NEUTRAL AMINO ACIDS BY SPIRODELA POLYRHIZA

SUMMARY Uptake of glycine, L-alanine, L-leucine, L-isoleucine, L-valine, and D-valine by intact fronds of the duckweed Spirodela polyrhiza was investigated. The time-course of amino acid uptake showed that the uptake rate (= net influx) was apparently not affected by accumulation of amino acid in the fronds, suggesting that there was no trans-inhibition of the influx, and that the efflux was negligible. Efflux was measured from fronds preloaded with L-(14C)leucine. When the external and internal concentration were both about 2.5 mM the efflux was ≤ 1% of the influx. Uptake rates were measured over a wide concentration range, 1 μM to 10 mM. For each amino acid the relation between uptake rate and external concentration could be described as the sum of two Michaelis-Menten processes. It is suggested that the duckweed possesses two transport systems common to the neutral amino acids tested: a low Km, low capacity system (system 1), and a high Km, high capacity system (system 2).

Diffusive Resistance, Titratable Acidity, and CO 2 Fixation in Two Tropical Epiphytic Ferns

Crassulacean acid metabolism (CAM) is known to occur in many succulent plant species (Ranson & Thomas 1960; Ting et al., 1972; Wolf, 1960). To date, more than 184 plant species have been reported to exhibit CAM features, but none of them are ferns (Szarek, pers. comm.). Recently we reported nocturnal assimilation of C02 by Drymoglossum piloselloides (L.) Presl, an epiphytic fern (Hew & Wong, 1974). This paper presents further evidence to support our previous findings that certain epiphytic ferns do exhibit characteristics of CAM plants. Two epiphytic ferns, Pyrrosia longifolia (Burm.) Morton and Drymoglossum piloselloides, were chosen for the present investigation. These ferns are found frequently on the lower part of Acacia tree trunks. Gleichenia linearis (Burm.) Clarke, a terrestial sun fern which has been shown to be a C3 plant (Hew & Wong, 1974), also was included in the study. All three ferns grow wild around the Nanyang University campus. For comparison purposes, the flowering plant Kalanchoe pinnata, a known CAM plant, was also used as experimental material. For 14C02 fixation studies and determination of titratable acidity, detached leaves or fronds were used; the method for 14C02 fixation has been described previously (Wong & Hew, 1973). Titratable acidity of plant tissues was determined as described by Szarek and Ting (1974), except the leaf or frond extract was titrated to a pH7 end point. Diffusive resistance of intact fronds or leaves were measured at three hour intervals using an Li-60 Diffusive Resistance Meter (LiCor Limited). Titratable Acidity.-The diurnal changes in titratable acidity of Drymoglossum, Pyrrosia, and Kalanchoe were similar (Figs. 1 -3). Titratable acidity decreased in the light, and at night the acidity increased. The magnitude of dark acidification in these three species was comparable to that previously reported (Bruinsma, 1958; McWilliams, 1970; Szarek & Ting, 1974). Among the three species, titratable acidity was highest in Kalanchoe, both in light and darkness. There was no significant difference in titratable acidity between the two ferns. In contrast, Gleichenia (Fig. 4), which is a C3 plant, shows no diurnal fluctuation in titratable acidity. Diffusive Resistance.-Changes in diffusive resistance patterns of intact Pyrrosia fronds (Fig. 5) and Kalanchoe leaves (Fig. 6) in the day and at night were similar, with high diffusive resistance in the day and low at night. The values for minimum diffusive resistance (5-15 secocm-1 ) and maximum diffusive resistance (100-120 sec*cm ) also were in agreement with that of other succulent plants (Szarek & Ting, 1974; Ting et al., 1972). From the changes in patterns, one could conclude that Pyrrosia and Drymoglossum stomata were closed in the day and open at night (Nishida, 1963; Ting et al., 1972). A point worth noting is that with the onset of darkness, an increase in diffusive resistance in both Pyrrosia and

Isolation and Propeties of Chloroplasts from the Siphonous Green Alga Cualerpa simpliciuscula

Isolated chloroplasts fixed CO*2 and evolved oxygen at rates equal to or greater than rates measured in segments of plant frond, with a maximum hourly value of 73 �mol per mg chlorophyll (a+ b). They were equally active whether isolated in a sorbitol-based medium or in an artificial sea water medium. Their activity declined after isolation, although they were relatively resistant to lysis in hypotonic medium. The products of photosynthesis included both sucrose and alanine as well as hexose monophosphates, and organic acids. There was much less 1,3-�-glucan formation in the chloroplasts than in the intact plant fronds. These chloroplasts appeared to be free of gross contamination when seen under the light microscope, but material which could not be distinguished from cytoplasm was attached to some chloroplasts. A maximum value of 17% contamination was calculated from the distribution of the mitochondrial marker enzyme succinate dehydrogenase.

TRANSLOCATION IN THE GIANT KELP MACROCYSTIS I. RATES, DIRECTION, QUANTITY OF C14‐LABELED PRODUCTS AND FLUORESCEIN

SUMMARY C14 tracer and fluorescein dye experiments on translocation through the stipes of Macrocystis are reported. Both dye and C14‐labeled products move mainly in the sieve tubes acropetally and basipetally in variable amounts and at variable rates, frequently entering young developing blades en route. The maximum rate of transport of organic products of photosynthesis may reach 65‐78 cm/hr, while that of fluorescein rarely exceeds 1.0 cm/hr in intact fronds. By their behavior, fluorescein and the C1J′‐labeled products(s) resemble phloem translocation in vascular plants.