DISSECTION OF TWO DISTINCT DEFENSE‐RELATED RESPONSES TO AGAR OLIGOSACCHARIDES IN GRACILARIA CHILENSIS (RHODOPHYTA) AND GRACILARIA CONFERTA (RHODOPHYTA)1

The two agar‐producing red algae, Gracilaria chilensis C. J. Bird, McLachlan & E. C. Oliveira and Gracilaria conferta (Schousboe ex Montagne) Montagne, responded with hydrogen peroxide (H₂O₂) release when agar oligosaccharides were added to the medium. In G. conferta, a transient release was observed, followed by a refractory state of 6 h. This response was sensitive to chemical inhibitors of NADPH oxidase, protein kinases, protein phosphatases, and calcium translocation in the cell, whereas it was insensitive to inhibitors of metalloenzymes. Transmission electron microscopic observations of the H₂O₂‐dependent formation of cerium peroxide from cerium chloride indicated oxygen activation at the plasma membrane of G. conferta. A putative system, consisting of a receptor specific to agar oligosaccharides and a plasma membrane‐located NADPH oxidase, appears to be responsible for the release of H₂O₂ in G. conferta. Subcellular examination of G. chilensis showed that the H₂O₂ release was located in the cell wall. It was sensitive to inhibitors of metalloenzymes and flavoenzymes, and no refractory state was observed. The release was correlated with accumulation of an aldehyde in the algal medium, suggesting that an agar oligosaccharide oxidase is present in the apoplast of G. chilensis. The presence of this enzyme could also be demonstrated by polyacrylamide electrophoresis under nondenaturating conditions and proven to be variable. Cultivation of G. chilensis at 16 to 17°C resulted in significantly stronger expression of agar oligosaccharide oxidase than cultivation at 12°C, which indicates that the enzyme is used under conditions that generally favor microbial agar macerating activity.     

DIURNAL FLUCTUATION OF NITRATE REDUCTASE ACTIVITY IN THE MARINE RED ALGA GRACILARIA TENUISTIPITATA (RHODOPHYTA)1

The biochemical characteristics and diurnal changes in activity of the enzyme nitrate reductase (NR; EC 1.6.6.1) from the marine red alga Gracilaria tenuistipitata var. liui Zhang et Xia are described. Different assay conditions were tested to determine the stability of NR. The crude extract of G. tenuistipitata has a NR specific activity of 10.2 U.mg⁻¹, which is higher than the NR activities found for other algae, plants, and fungi. This NR is highly active at a slightly alkaline pH and is stable over a wide range of temperature, with an optimal activity at 20° C. The apical portions of the thallus contain 64.9 ± 6.6% of the total NR specific activity. The apparent Michaelis-Menten (K_m) constant found for KNO₃ was 197 μM, and it was 95 μM for NADH. The NR from G. tenuistipitata can be included in the NADH-specific group, because no activity was found when NADPH was used as an electron donor. In extracts of algae grown under either continuously dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at the middle of the light phase, when activity is 30-fold higher than during the night phase.     

RESPONSE OF GRACILARIA CONFERTA (RHODOPHYTA) TO OLIGOAGARS RESULTS IN DEFENSE AGAINST AGAR-DEGRADING EPIPHYTES

Elicitation of Gracilaria conferta (Schousboe ex Montagne) J. et G. Feldmann with oligoagars resulted in a defense response that was strong enough to kill epiphytic bacteria associated with the alga. Up to 60% of the resident bacterial flora of healthy plants was eliminated within 60 min after addition of neoagarohexaose to the algal medium. Single isolates of agar-degrading bacteria that had been isolated previously from healthy or decaying algal tissues proved to be more sensitive. Some of them were generally unable to survive on healthy G. conferta. Others survived on unelicited plants. Approximately 90% of these more resistant agar degraders were eliminated within 15 min after elicitation. The bacterial degradation of dead tissue of G. conferta resulted in a release of elicitors. The elicitors accumulated in the medium and reached high enough concentrations within 24 h to induce a hypersensitive response in healthy algae. The eliciting agent could be destroyed with β-agarase and was thus probably oligoagar. Application of antibiotics prevented the accumulation of the elicitor, which indicated that bacteria were responsible for its release from the algal biomass. The hypersensitive response of G. conferta after contact with oligoagars is thus a true defense response, because it enables the plant to protect itself efficiently from enzymatic attacks on its cell wall.     

THE EFFECTS OF LIGHT AND NITROGEN ON GROWTH,PIGMENT CONTENT,AND BIOCHEMICAL COMPOSITION OF GRACILARIA FOLIIFERA V. ANGUSTISSIMA (GIGARTINALES,RHODOPHYTA)1

The combined effects of light intensity and nitrogen (NO₃^?) on growth rate, pigment content, and biochemical composition of Gracilaria foliifera v. angustissima (Harvey) Taylor was investigated using outdoor continuous cultures. Growth of Gracilaria increased linearly with increasing light to 0.43 doublings d^?1 at high light levels (383 ly d^?1 of in situ light), suggesting that light may often limit growth of this plant in nature. Chlorophyll a and phycoerythrin contents were inversely proportional to light level and growth rate. However, pigment content did not affect the growth capacity of Gracilaria. There was no increase in growth or pigment content with increasing additions of nitrogen. The low nitrogen treatment was unenriched seawater that had higher NO₃^? levels than most coastal waters (influent = 8.61 μM; residual = 0.94 μM). When growing near its maximum rate under high light intensities, Gracilaria had a significantly (P < 0.001) lower phycoerythrin: chlorophyll a ratio (phyco: Chl a) than did Gracilaria growing more slowly under lower light (Phyco:Chl a of 2.8 ± 0.2 vs. 3.8 ± 0.3). Faster growing plants also had C:N ratios above 10, indicating N- limitation. In addition to harvesting light the phycobiliproteins of Gracilaria may store nitrogen. Growth rates of Gracilaria correlated negatively with ash (r =–0.85) and positively with the carbon: phycoerythrin ratio (r = 0.85), suggesting that these two indices can be used to estimate growth in the field.     

SPORELING COALESCENCE AND INTRACLONAL VARIATION IN GRACILARIA CHILENSIS (GRACILARIALES,RHODOPHYTA)1

This study evaluates the hypothesis that spore coalescence may cause intraclonal variation. Spore coalescence might allow the occurrence of unitary thalli that in fact correspond to genetically different, coalesced individuals. Plant portions simultaneously derived from these chimeric individuals may exhibit dissimilar growth responses even when incubated under similar abiotic conditions. Testing of the hypothesis included various approaches. Transmission electron microscopy observations of early stages of sporeling coalescence indicated that polysporic plantlets were formed by groups of spores and their derivatives. Even though adjacent cells in two different groups may fuse, these groups maintained an independent capacity to grow and form uprights. Laboratory-grown plantlets showed a significant correlation between the initial number of spores and the total number of erect axes differentiated from the sporeling. Construction and growth of bicolor individuals indicated the chimeric nature of the coalesced individuals. Coalesced, bicolor holdfasts had green and red cells, which subsequently produced green and red uprights, respectively. Individuals fronds were also chimeric, as indicated by the production of green and red branchlets from single, red uprights. The existence of mixed tissues was further substantiated by random amplified polymorphic DNA analysis. The banding pattern produced by branchlets of a unisporic thallus was consistently monomorphic, whereas the patterns produced by the polysporic thallus were polymorphic. Growth rates of polysporic thalli had larger data dispersal and variation coefficients than oligosporic or monosporic thalli. Therefore, all results support the original hypothesis and suggest that coalescence might be ecologically more important than previously thought.     

MOLECULAR IDENTIFICATION OF TWO SIBLING SPECIES UNDER THE NAME GRACILARIA CHILENSIS (RHODOPHYTA,GRACILARIALES)1,3

Molecular markers belonging to three different genomes, mitochondrial (cox2‐3 spacer), plastid (RUBISCO spacer), and nuclear (internal transcribed spacer 1), were used to compare Gracilaria chilensis samples collected along the Chilean coast with samples ascribed to G. chilensis from the West Pacific Ocean (New Zealand and Australia). Our data are in agreement with previous studies suggesting two sibling species currently going under the name G. chilensis that co‐occur in New Zealand. One of these, a New Zealand sample previously examined by Bird and others in 1990, is conspecific with G. chilensis from Chile. Finally, our results demonstrate clearly that most of the sequences in GenBank reported as G. chilensis are based on misidentified material.     

ROLE OF PROLINE ACCUMULATION IN RESPONSE TO TOXIC COPPER IN CHLORELLA SP. (CHLOROPHYCEAE) CELLS

The green alga Chlorella sp. (strain 2350) was found to accumulate proline, up to a maximum of 0.027 fmol·cell⁻¹, under stressful concentrations of cupric ions. The function of the accumulated proline was studied with respect to its effect on copper (Cu) uptake. By induction with salt stress, cells with various levels of intracellular proline were used for this study. It is shown that the amount of Cu taken up by the cells was reduced when the intracellular proline levels were enhanced. When proline was exogenously supplied prior to Cu treatment, the adsorption of Cu was markedly reduced. When exogenous proline was supplied after Cu treatment, it resulted in a remarkable desorption of the adsorbed Cu immediately after the addition of proline. The results of the present study indicate that proline may exert some action on the cell surface and suggest that one function of accumulated proline is to reduce the uptake of metal ions. The accumulation of proline may be related to a tolerance mechanism for dealing with Cu stress.     

ENHANCEMENT OF INTRACELLULAR PROLINE LEVEL IN CELLS OF ANACYSTIS NIDULANS (CYANOBACTERIA) EXPOSED TO DELETERIOUS CONCENTRATIONS OF COPPER1

The intracellular proline level in Anacystis nidulans cells was enhanced when the cells were exposed to sublethal concentrations of Cu²⁺; the degree of enhancement was positively related to the concentration of Cu²⁺. Analysis by high-performance liquid chromatography confirmed that the enhancement of proline levels was the most pronounced change in the composition of the free amino acid pool during copper treatment. A direct supply of exogenous proline to the cultures lowered the inhibitory influence of Cu²⁺on the growth of cells. Further experiments showed that the supply of exogenous proline lowered the leakage of potassium ions from cells exposed to deleterious concentrations of Cu²⁺. The inhibition of potassium leakage was particularly pronounced when proline was supplied prior to Cu²⁺treatment. The present study suggests that enhanced proline protects cell membranes from being affected by deleterious concentrations of Cu²⁺.     

LOW-TEMPERATURE-INDUCED SYNTHESIS OF α-CAROTENE IN THE MICROALGA DUNALIELLA SALINA (CHLOROPHYTA)

The microalga Dunaliella salina (Teo.) is well known as an accumulator of β-carotene (β,β-carotene) when subjected to growth-limiting conditions (e.g. exposure to high irradiances). In addition, the carotenoid α-carotene (β,ε-carotene) may also be synthesized and subsequently accumulated by this alga under specific growth conditions. The main factor in stimulating the synthesis of this carotene was determined to be exposure to lower than optimum temperatures for algal growth. A 7.5-fold increase in the levels of α-carotene was observed when the temperature was decreased from 34 to 17° C, whilst levels of β-carotene were unaltered. The accumulation of α-carotene was unaffected by irradiance, although its isomeric composition was greatly altered by light levels. The proportion of 9- cis α-carotene increased from 15% to 45% of total α-carotene when the irradiance was decreased from 260 to 50 μmol·m⁻²·s⁻¹. Exposure to higher irradiances had little influence on the isomeric composition of this carotenoid. A reduction in growth temperature did not influence the isomeric composition of α-carotene. Nutrient status (nitrogen and phosphate) had no effect on either the content or isomeric composition of α-carotene accumulated by D. salina.     

GEOGRAPHIC VARIATION IN THERMAL TRAITS IN DIGENEA SIMPLEX AND CHAMPIA PARVULA (RHODOPHYTA) IN RELATION TO PRESENT AND GLACIAL TEMPERATURE REGIMES

Geographic variation in temperature responses (survival and growth) was investigated in two red algae: Digenea simplex (Wulfen) C. Agardh and Champia parvula (C. Agardh) Harvey. D. simplex has a tropical to warm temperate distribution; C. parvula extends from the tropics into the cold temperate zone. Ecoclinal variation was found in both species but was much stronger in C. parvula than in D. simplex. The former species showed variation in upper and lower tolerance limits as well as in the upper and lower limits for growth. The latter species showed variation mainly in its lower tolerance limit. Ecoclinal variation was related to the amount of present and glacial selection pressure along the climate gradient. In both species, isolates from the colder localities had insufficient cold tolerance to have survived low glacial winter temperatures, so these locations must have been colonized after the end of the glaciation. Eastern Mediterranean and Atlantic populations were probably isolated during the glaciation by a thermal barrier at the entrance of the Mediterranean. In C. parvula, evidence existed for a trade-off between the performance at high and at low temperatures, which would enhance selection pressure in opposite directions at either end of the climatic range. No evidence for such a trade-off was found in D. simplex.     

PHOTOSYNTHESIS OF THE RED ALGA GASTROCLONIUM COULTERI (RHODOPHYTA) IN RESPONSE TO CHANGES IN TEMPERATURE,LIGHT INTENSITY,AND DESICCATION1

Previously reported transplantation experiments in the field showed that Gastroclonium coulteri (Harvey) Kylin could survive above its normal intertidal range (0.0–0.5 m above MLLW), except during periods of daytime low tides in spring. Net photosynthetic rate measurements in the laboratory were performed to determine which physical factors might determine the upper boundary for this species in the intertidal zone. Maximum net photosynthesis occurred between 15 and 20° C, but remained positive between 4 and 35° C. The air temperature extremes observed in the field were 2° C (only seen once) and 26° C. Net photosynthesis increased as expected with light intensity to the highest value obtainable in the laboratory, 1400 μEin m^?2 s^?1. Plants collected from the field under higher light intensity (up to 2000 μEin m^?2 s^?2) also showed high rates of photosynthesis. Neither the temperature nor light levels observed in the field were directly damaging to photosynthesis. Desiccation, however, resulted in a sharp decrease in both photosynthesis and respiration. G. coulteri fully recovered from successive daily treatments of about 35% desiccation, but not from successive treatments of 50% desiccation. One exposure to 70% desiccation allowed no recovery of photosynthetic capacity.     

DEFENSE EVOLUTION IN THE GRACILARIACEAE (RHODOPHYTA): SUBSTRATE‐REGULATED OXIDATION OF AGAR OLIGOSACCHARIDES IS MORE ANCIENT THAN THE OLIGOAGAR‐ACTIVATED OXIDATIVE BURST1

Combined phylogenetic, physiological, and biochemical approaches revealed that differences in defense‐related responses among 17 species belonging to the Gracilariaceae were consistent with their evolutionary history. An oxidative burst response resulting from activation of NADPH oxidase was always observed in two of the subgenera of Gracilaria sensu lato (Gracilaria, Hydropuntia), but not in Gracilariopsis and in species related to Gracilaria chilensis (“chilensis” clade). On the other hand, all species examined except Gracilaria tenuistipitata var. liui and Gracilariopsis longissima responded with up‐regulation of agar oligosaccharide oxidase to an challenge with agar oligosaccharides. As indicated by pharmacological experiments conducted with Gracilaria chilensis and Gracilaria sp. “dura,” the up‐regulation of agar oligosaccharide oxidase involved an NAD(P)H‐dependent signaling pathway, but not kinase activity. By contrast, the activation of NADPH oxidase requires protein phosphorylation. Both responses are therefore independent, and the agar oligosaccharide‐activated oxidative burst evolved after the capacity to oxidize agar oligosaccharide, probably providing additional defensive capacity to the most recently differentiated clades of Gracilariaceae. As demonstrated with Gracilaria gracilis, Gracilaria dura, and Gracilariopsis longissima, the different responses to agar oligosaccharides allow for a fast and nondestructive distinction among different clades of gracilarioids that are morphologically convergent. Based upon sequences of the chloroplast‐encoded rbcL gene, this study suggests that at least some of the samples from NW America recorded as Gs. lemanaeiformis are probably Gs. chorda. Moreover, previous records of Gracilaria conferta from Israel are shown to be based upon misidentification of Gracilaria sp. “dura,” a species that belongs to the Hydropuntia subgenus.     

THE ROLE OF NITROGEN NUTRITION IN HIGH-TEMPERATURE TOLERANCE OF THE KELP,LAMINARIA SACCHARINA (CHROMOPHYTA)1

Mechanisms of high-temperature tolerance in the kelp Laminaria saccharina (L.) Lamour. were examined by comparing a heat-tolerant ecotype from Long Island Sound (LIS), New York, and a population from the Atlantic (ATL) coast of Maine. Greater heat tolerance was not attributable to greater thermal stability of the photosynthetic apparatus: LIS and ATL plants exhibited similar short-term effects of high temperature on photosynthetic capacity (P_max) and quantum yield (estimated as the ratio of variable to maximum chlorophyll fluorescence, F_v/F_m. As LIS plants had consistently higher N and protein content than ATL plants, the interaction between nitrogen nutrition and high-temperature tolerance was examined. When grown under high N supply and optimal temperature (12° C), LIS plants had a higher density of photosystem II reaction centers (RCII), higher activity of two Calvin cycle enzymes (ribulose bisphosphate carboxylase oxygenase [RUBISCO] and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase [G3PDH]), and higher P_max and F_v/F_m than ATL plants. Individual ATL plants, furthermore, exhibited close correlations of RCII density and enzyme activity with N and/or protein content. Variation in RCII density and enzyme activity, in turn, largely accounted for plant-to-plant differences in P_max and F_v/F_m. Relationships among these parameters were generally weak or lacking among individual LIS plants grown under optimal conditions, apparently because luxury N consumption resulted in excess reserves of photosynthetic apparatus components. Exposure of N-replete LIS and ATL plants to a superoptimal temperature (22° C) for 4 days caused an increase in the minimum turnover time of the photosynthetic apparatus (tau) and a decrease in P_max, but had no consistent effect on F_v/F_m RCII density, PSU size (chlorophyll a/RCII), or enzyme activities. When plants were subjected to concurrent N limitation and heat stress, however, LIS and ATL populations exhibited quite different responses. All photosynthetic parameters of N-limited ATL plants declined sharply in response to high temperature, resulting in a negative rate of daily net C fixation. In contrast, LIS plants showed a reduction in PSU size, but maintained other parameters, including daily C fixation, at levels similar to those of N-limited plants at optimal temperature. Overall, the ability of LIS plants to accumulate and maintain high N reserves appears to be critical for heat tolerance and, therefore, for survival during summer periods of simultaneous low N supply and superoptimal temperature. ATL plants, which also experience low summer N supply but not superoptimal temperatures, do not accumulate large reserves of nitrogenous components and are unable to tolerate the combined stress. Because low N supply often co-occurs with high temperatures in temperate marine systems, large-scale declines in algal productivity, such as during El Niño events, are probably due to the interactive effect of N limitation and heat stress.     

菠萝叶膜脂脂肪酸组成的温度效应及其与抗寒性的关系

生长在不同季节的菠萝叶膜脂脂肪酸的配比存在着明显差异;随着大气温度的下降,18:1含量显著减少,18:2和18:3含量增加。不同品种均表现出一致的变化趋势。致害低温破坏了膜脂,使较不抗寒品种的16:0含量增加,18:2和18:3含量减少;较抗寒品种这种变化则较不显著。适当低温锻炼能改变膜脂脂肪酸的代谢过程,16:0和18:1含量减少,18:3含量增加。当处于更低温度时,除了16:0和18:1继续减少外,有一部分18:2也脱饱和而转变为18:3。因之明显地增加了膜脂中18:3的含量和脂肪酸的不饱和度,从而有利于抗寒性的提高。而品种间的抗寒性差异亦是在此低温期间表现出来。     

高温对天蚕卵黄发生的影响(英文)

３２℃高温对天蚕（Ａｎｔｈｅｒａｅａ ｙａｍａｍａｉ）卵黄发生影响明显,其中影响程度与开始经受３２℃处理时天蚕发育阶段有关。结茧后第１天开始即受３２℃处理时,脂肪体和血淋巴中卵黄原蛋白（Ｖｇ）与可溶性蛋白含量均低于受２６℃处理的;化蛹后第１天开始即受３２℃处理时,脂肪体中Ｖｇ滴度与２６℃下的基本无明显差异,但血淋巴和卵巢中Ｖｇ或卵黄磷蛋白含量（Ｖｔ）均低于２６℃的;而在化蛹后第６天开始受３２℃处理时,脂肪体和血淋巴中Ｖｇ含量与２６℃的无明显差异,卵巢中Ｖｔ则明显高于２６℃的。此外,曾就化蛹后第１或６天开始受３２℃处理后脂肪体、血淋巴和卵巢中可溶性蛋白含量的变化作了检测。建议在饲育或保护制种用茧蚕时,老熟幼虫和化蛹初期的蚕蛹不宜接触３２℃高温。