MOLECULAR AND MORPHOLOGICAL ANALYSIS OF ENTEROMORPHA INTESTINALIS AND E. COMPRESSA (CHLOROPHYTA) IN THE BRITISH ISLES

The very common green seaweeds Enteromorpha intestinalis (L.) Nees and E. compressa (L.) Nees are important fouling organisms and have commonly been used as indicators of eutrophication, but their taxonomic status is problematic. The genus presents extreme difficulties because there is wide intraspecific variation in morphology, but morphological differences between species are small and difficult to detect. In this study, molecular data were used in parallel with morphological characters to resolve the taxonomic problems. Phylogenetic analysis of sequences of the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene distinguished two groups of samples, which were identified by morphological characters as E. compressa (branched) and E. intestinalis (normally unbranched). There was a low level of sequence divergence within each group of samples, but divergence between groups was as great as that between either of the two species and the outgroup E. prolifera. Clades representing E. compressa and E. intestinalis were also found in analyses of an independent molecular data set, chloroplast DNA restriction fragment length polymorphisms (RFLPs). Enteromorpha intestinalis and E. compressa represent two distinct, genetically divergent species. Reinterpretation of published studies shows that these species are reproductively isolated. However, E. compressa and E. intestinalis are sometimes very difficult to distinguish from each other and could be regarded as cryptic species. The presence or absence of branching was the most useful character distinguishing these two species, but there was an element of ambiguity because low salinity or salinity shock can induce branching in E. intestinalis. If environmental factors such as salinity are taken into account, branching can be used to identify the great majority of thalli correctly. This study therefore provides a basis for identifying the two most important marine fouling macroalgae and for their use in environmental monitoring and experimentation. Typification of these two Linnaean species showed that current usage of the names accords with the lectotype and protologue of both species. Samples that resembled E. usneoides did not form a clade in any of the trees, and constraining the data to support the monophyly of this group incurred a penalty. Enteromorpha usneoides appears to be an ecotype of E. compressa.     

PHOTOSYNTHETIC CARBON METABOLISM IN ENTEROMORPHA COMPRESSA (CHLOROPHYTA)1

The intertidal macroalga Enteromorpha compressa showed the ability to use HCO₃^? as an exogenous inorganic carbon (Ci) source for photosynthesis. However, although the natural seawater concentration of this carbon form was saturating, additional CO₂ above ambient Ci levels doubled net photosynthetic rates. Therefore, the productivity of this alga, when submerged, is likely to be limited by Ci. When plants were exposed to air, photosynthetic rates saturated at air-levels of CO₂ during mild desiccation. Based on carbon fixing enzyme activities and Ci pulsechase incorporation patterns, it was found that Enteromorpha is a C₃ plant. However, this alga did not show O₂ inhibited photosynthetic rates at natural seawater Ci conditions. It is suggested that such a C₄- like gas exchange response is due to the HCO₃^? utilization system concentrating CO₂ intracellularly, thus alleviating apparent photorespiration.     

The role of lateral roots in bypass flow in rice (Oryza sativa L.)

Although an apoplastic pathway (the so‐called bypass flow) is implicated in the uptake of Na⁺ by rice growing in saline conditions, the point of entry of this flow into roots remains to be elucidated. We investigated the role of lateral roots in bypass flow using the tracer trisodium‐8‐hydroxy‐1,3,6‐pyrenetrisulphonic acid (PTS) and the rice cv. IR36. PTS was identified in the vascular tissue of lateral roots using both epifluorescence microscopy and confocal laser scanning microscopy. Cryo‐scanning electron microscopy and epifluorescence microscopy of sections stained with berberine‐aniline blue revealed that the exodermis is absent in the lateral roots. We conclude that PTS can move freely through the cortical layers of lateral roots, enter the stele and be transported to the shoot via the transpiration stream.     

EFFECT OF NITROGEN AND PHOSPHORUS SUPPLY ON GROWTH AND TISSUE COMPOSITION OF ULVA FENESTRATA AND ENTEROMORPHA INTESTINALIS (ULVALES,CHLOROPHYTA)1

The chlorophyte macroalgae Ulva fenestrata (Postels and Ruprecht) and Enteromorpha intestinalis (Linnaeus) Link. were grown under various nutrient regimes in indoor semi-continuous and batch cultures. Tissue nitrogen contents ranged from 1.3–5.4% N (dry wt), whereas tissue P ranged from 0.21–0.56% P (dry wt). Growth in low nitrogen medium resulted in N:P ratios of 5–8, whereas growth in high nitrogen medium resulted in N:P ratios of 21–44. For U. fenestrata, tissue N:P < 16 was indicative of N-limitation. Tissue N:P 16–24 was optimal for growth and tissue N:P > 24 was indicative of P-limitation. Growth of U. fenestrata was hyperbolically related to tissue N but linearly related to tissue P. Phosphorus-limited U. fenestrata maintained high levels of tissue N, but N-limited algae became depleted of P. For E. intestinalis, tissue N remained at maximum levels during P-limitation whereas tissue P decreased to about 85% of maximal levels during N-limitation. Growth rates for U. fenestrata decreased faster during P-limitation than during N-limitation. Simultaneously, tissue P was depleted faster than tissue N. Our results suggest that comparing tissue N and P of macroalage grown in batch cultures is useful for monitoring the nutritional status of macroalgae.     

细胞外钠离子浓度对羊浦肯野纤维膜钠泵活动的影响

采用离子选择电极测量羊浦肯野纤维细胞膜内钠离子活度(~(ai)N_a),细胞间钾离子活度(a~ok)及细胞膜电位(v_m),观察不同浓度低钠,无钙液对其影响,在无钙低钠液中,细胞内Na~+逐出,α~iNa 降低,其变化速率,幅值与[Na]_o 相关,同时也受细胞 a~iNa 初始水平(aiNa(o))的影响。aiNa 下降6min 时的稳态水平与[Na]_o 呈直线正相关,这些结果表明,[Na]_o 降低时,细胞膜钠泵活动加强,细胞内 Na~+逐出增加,其最终结果是使 Na+跨膜梯度维持相对稳定,因而可以认为是 Na~+跨膜梯度而不是单纯的细胞内 Na~+控制膜钠泵活动。在低 Na~+液引起细胞内 Na~+主动逐出增加的同时,细胞膜出现超极化,[Na]_o 愈低,膜超极化程度愈高,从低钠液引起的 a~i_(Na),V_m,α~o_k 变化之间的时程关系看,膜超极化主要由加大的外向泵电流引起,同时发生的细胞间 K~+浓度变化对其也有一定影响。     

EXTENSIVE INTRASPECIFIC MORPHOLOGICAL VARIATION IN ENTEROMORPHA MUSCOIDES (CHLOROPHYTA) REVEALED BY MOLECULAR ANALYSIS

Enteromorpha muscoides (Clemente y Rubio) Cremades and E. clathrata Roth (Greville) are morphologically variable species that can easily be distinguished from other Enteromorpha species but not from each other. The key morphological character separating the two species is the presence or absence of spine-like branches: E. muscoides has small spine-like branches throughout the thallus, whereas E. clathrata lacks spines. The spiny branches in E. muscoides are not as obvious in summer as in winter, so summer samples may be difficult to distinguish from those of E. clathrata. In this study, molecular data were used to investigate whether these two species, as defined by morphological characters, might be conspecific. The sequences of the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene differed by 0%–0.6% between all samples of both E. muscoides and E. clathrata. Phylogenetic analysis of these sequences in an alignment with 13 other representatives of both Enteromorpha and Ulva showed that this highly supported monophyletic E. muscoides / E. clathrata clade is separated by long branch lengths from other Enteromorpha and Ulva clades. Based on these results, we suggest that Enteromorpha muscoides (Clemente y Rubio) Cremades and Enteromorpha clathrata Roth (Greville) are conspecific, with the older name E. muscoides taking priority.     

CONTRASTING EFFECTS OF METHIONINE SULFOXIMINE ON UPTAKE AND ASSIMILATION OF AMMONIUM IN ULVA INTESTINALIS (CHLOROPHYCEAE)1

Ammonium is assimilated in algae by the glutamine synthetase (GS)–glutamine:2‐oxoglutarate aminotransferase pathway. In addition to the assimilation of external ammonium taken up across the cell membrane, an alga may have to reassimilate ammonium derived from endogenous sources (i.e. nitrate reduction, photorespiration, and amino acid degradation). Methionine sulfoximine (MSX), an irreversible inhibitor of GS, completely inhibited GS activity in Ulva intestinalis L. after 12 h. However, assimilation of externally derived ammonium was completely inhibited after only 1–2 h in the presence of MSX and was followed by production of endogenous ammonium. However, endogenous ammonium production in U. intestinalis represented only a mean of 4% of total assimilation attributable to GS. The internally controlled rate of ammonium uptake (V_i) was almost completely inhibited in the presence of MSX, suggesting that V_i is a measure of the maximum rate of ammonium assimilation. After complete inhibition of ammonium assimilation in the presence of MSX, the initial or surge (V_s) rate of ammonium uptake in the presence of 400 μM ammonium chloride decreased by only 17%. However, the amount that the rate of ammonium uptake decreased by was very similar to the uninhibited rate of ammonium assimilation. In addition, the decrease in the rate of ammonium uptake in darkness (in the absence of MSX) in the presence of 400 μM ammonium chloride matched the decrease in the rate of ammonium assimilation. However, in the presence of 10 μM ammonium chloride, MSX completely inhibited ammonium assimilation but had no effect on the rate of uptake.     

GROWTH-INHIBITORS AND GROWTH-PROMOTERS IN ENTEROMORPHA COMPRESS A (CHLOROPHYTA)1

Methanol extracts from the alga Enteromorpha compressa (L.) Grev. contain substances which inhibit the elongation of Lepidium roots. Chromatographic separation of the inhibiting substances revealed that one of the inhibitory zones of the chromatograms had properties of the so-called inhibitor β. Neither abscisic acid (ABA) nor lunularic acid proved to be responsible for the growth-inhibiting property of this zone. Moreover, the extracts contain substances which promote the elongation of Avena coleoptile segments. One of these substances could be tentatively identified as indole-3-acetic acid by thin-layer and gas-liquid chromatography. (In addition to indole-3-acetic acid a second growth-promoting factor with the properties of the so-called accelerator α could be detected.)     

A new screening technique for salinity resistance in rice (Oryza sativa L.) seedlings using bypass flow

A lack of screening techniques delays progress in research on salinity resistance in rice. In this study, we report our test of the hypothesis that an apoplastic pathway (the so-called bypass flow) causes a difference in salt resistance between rice genotypes and can be used in screening for salinity resistance. Fourteen-day-old seedlings of low- and high-Na(+) -transporting recombinant inbred lines (10 of each) of rice IR55178 were treated with 50 mm NaCl and 0.2 mm trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS), a bypass flow tracer, for short (4 d) and long (90 d) periods of time. The results showed that the average shoot Na(+) concentration and bypass flow for high-Na(+) -transporting lines were 1.4 and 2.4 times higher than those of low-Na(+) -transporting lines, respectively. There was a positive linear correlation between the percentage of bypass flow and Na(+) concentrations in the shoots, suggesting that the difference in Na(+) transport in rice is a consequence of different degrees of bypass flow. Moreover, a high correlation was found between bypass flow and seedling survival after prolonged salt stress: the lower the magnitude of bypass flow, the greater the seedling survival. We conclude that bypass flow could be used as a new screening technique for salt resistance in rice.     

DIURNAL CELL DIVISION REGULATED BY GATING THE G1/S TRANSITION IN ENTEROMORPHA COMPRESSA (CHLOROPHYTA)1

The cell‐cycle progression of Enteromorpha compressa (L.) Nees (=Ulva compressa L.) was diurnally regulated by gating the G₁/S transition. When the gate was open, the cells were able to divide if they had attained a sufficient size. However, the cells were not able to divide while the gate was closed, even if the cells had attained sufficient size. The diurnal rhythm of cell division immediately disappeared when the thalli were transferred to continuous light or darkness. When the thalli were transferred to a shifted photoperiod, the rhythm of cell division immediately and accurately synchronized with the shifted photoperiod. These data support a gating‐system model regulated by light:dark (L:D) cycles rather than an endogenous circadian clock. A dark phase of 6 h or longer was essential for gate closing, and a light phase of 14 h was required to renew cell division after a dark phase of >6 h.     

Salt tolerance of maize (Zea mays L.): the role of sodium exclusion

The influence of NaCl and Na₂SO₄ on growth of two maize cultivars (Zea mays cv. Pioneer 3906 and cv. Across 8023) differing in Na⁺ uptake was investigated in two green-house experiments. Na⁺ treatment with different accompanying anions (Cl^?/SO₄^2?) showed that ion toxicity was caused by Na⁺. While shoot growth of the two cultivars was markedly affected by salt in comparison to the control during the first 2–3 weeks, there were only slight differences between the cultivars. The shoot Ca²⁺ concentration was reduced in both cultivars, and the youngest leaves contained an even lower concentration compared with the rest of the shoot. During this first phase, Across 8023 tended to have higher concentrations of Ca²⁺ than Pioneer 3906. The Na⁺-excluding cultivar Pioneer 3906 showed continuous, although reduced, growth compared with the control, while the Na⁺ concentration in the shoot decreased until flowering. Cultivar Across 8023 accumulated Na⁺ until flowering: the reduction in the growth of stressed plants was greater than that for Pioneer 3906. Leaves of cultivar Across 8023 showed clear toxic symptoms, while those of the more salt-tolerant cultivar Pioneer 3906 did not. It is concluded that Na⁺ exclusion contributes to the salt tolerance of maize.     

KINETICS OF AMMONIUM ASSIMILATION IN TWO SEAWEEDS, ENTEROMORPHA SP. (CHLOROPHYCEAE) AND OSMUNDARIA COLENSOI (RHODOPHYCEAE)

The kinetics of ammonium assimilation were investigated in two seaweeds from northeastern New Zealand, Enteromorpha sp. (Chlorophyceae, Ulvales) and Osmundaria colensoi (Hook. f. et Harvey) R.E. Norris (Rhodophyceae, Ceramiales), with the use of a recently developed method for measuring assimilation. In contrast to ammonium uptake, which was nonsaturable, ammonium assimilation exhibited Michaelis–Menten kinetics in both species. Maximum rates of assimilation (V_max) were 27 and 12 μmol·(g DW)⁻¹·h⁻¹ for Enteromorpha sp. and O. colensoi, respectively, with half-saturation (K_m) constants for assimilation of 18 and 41 μM. At environmentally relevant concentrations, assimilation accounted for all of the ammonium taken up by both species. The maximum rate of assimilation in Enteromorpha sp. resembled very closely that of the ammonium assimilatory enzyme, glutamine synthetase, when activities of the latter were measured in the presence of subsaturating substrate (glutamate and ATP) concentrations. Moreover, the initial rate of glutamine production (measured with HPLC) following ammonium enrichment was almost identical to the rates determined above. The rate of ammonium assimilation was therefore determined by three independent methods, two of which involve in vivo measurements, and it is suggested that the use of assimilation kinetics may be useful when examining the nutrient relations of seaweeds.