Search

Sacoglossan sea slugs offer fascinating systems to study the onset and persistence of algal-plastid symbioses. Elysia chlorotica is particularly noteworthy because it can survive for months, relying solely on energy produced by ingested plastids of the stramenopile alga Vaucheria litorea that are sequestered in cells lining its digestive diverticula. How this animal can maintain the actively photosynthesizing organelles without replenishment of proteins from the lost algal nucleus remains unknown. Here, we used RNA-Seq analysis to test the idea that plastid sequestration leaves a significant signature on host gene expression during E. chlorotica development. Our results support this hypothesis and show that upon exposure to and ingestion of V. litorea plastids, genes involved in microbe-associated molecular patterns and oxidative stress-response mechanisms are significantly up-regulated. Interestingly, our results with E. chlorotica mirror those found with corals that maintain dinoflagellates as intact cells in symbiosomes, suggesting parallels between these animal-algal symbiotic interactions.

Some sea slugs are capable of retaining functional sequestered chloroplasts (kleptoplasts) for variable periods of time. The mechanisms supporting the maintenance of these organelles in animal hosts are still largely unknown. Non-photochemical quenching (NPQ) and the occurrence of a xanthophyll cycle were investigated in the sea slugs Elysia viridis and E. chlorotica using chlorophyll fluorescence measurements and pigment analysis. The photoprotective capacity of kleptoplasts was compared to that observed in their respective algal source, Codium tomentosum and Vaucheria litorea. A functional xanthophyll cycle and a rapidly reversible NPQ component were found in V. litorea and E. chlorotica but not in C. tomentosum and E. viridis. To our knowledge, this is the first report of the absence of a functional xanthophyll cycle in a green macroalgae. The absence of a functional xanthophyll cycle in C. tomentosum could contribute to the premature loss of photosynthetic activity and relatively short-term retention of kleptoplasts in E. viridis. On the contrary, E. chlorotica displays one of the longest functional examples of kleptoplasty known so far. We speculate that different efficiencies of photoprotection and repair mechanisms of algal food sources play a role in the longevity of photosynthetic activity in kleptoplasts retained by sea slugs.

Plants in terrestrial systems have evolved in direct association with microbes functioning as both agonists and antagonists of plant fitness and adaptability. As such, investigations that segregate plants and microbes provide only a limited scope of the biotic interactions that dictate plant community structure and composition in natural systems. Invasive plants provide an excellent working model to compare and contrast the effects of microbial communities associated with natural plant populations on plant fitness, adaptation, and fecundity. The last decade of DNA sequencing technology advancements opened the door to microbial community analysis, which has led to an increased awareness of the importance of an organism's microbiome and the disease states associated with microbiome shifts. Employing microbiome analysis to study the symbiotic networks associated with invasive plants will help us to understand what microorganisms contribute to plant fitness in natural systems, how different soil microbial communities impact plant fitness and adaptability, specificity of host-microbe interactions in natural plant populations, and the selective pressures that dictate the structure of above-ground and below-ground biotic communities. This review discusses recent advances in invasive plant biology that have resulted from microbiome analyses as well as the microbial factors that direct plant fitness and adaptability in natural systems.

Competing Interests: The authors have declared that no competing interests exist.

The establishment of kleptoplasty (retention of "stolen plastids") in the digestive tissue of the sacoglossan Elysia chlorotica Gould was investigated using transmission electron microscopy. Cellular processes occurring during the initial exposure to plastids were observed in laboratory raised animals ranging from 1-14 days post metamorphosis (dpm). These observations revealed an abundance of lipid droplets (LDs) correlating to plastid abundance. Starvation of animals resulted in LD and plastid decay in animals <5 dpm that had not yet achieved permanent kleptoplasty. Animals allowed to feed on algal prey (Vaucheria litorea C. Agardh) for 7 d or greater retained stable plastids resistant to cellular breakdown. Lipid analysis of algal and animal samples supports that these accumulating LDs may be of plastid origin, as the often algal-derived 20∶5 eicosapentaenoic acid was found in high abundance in the animal tissue. Subsequent culturing of animals in dark conditions revealed a reduced ability to establish permanent kleptoplasty in the absence of photosynthetic processes, coupled with increased mortality. Together, these data support an important role of photosynthetic lipid production in establishing and stabilizing this unique animal kleptoplasty.

The soil microbial community acts as a reservoir of microbes that directly influences the structure and composition of the aboveground plant community, promotes plant growth, increases stress tolerance and mediates local patterns of nutrient cycling. Direct interactions between plants and rhizosphere-dwelling microorganisms occur at, or near, the surface of the root. Upon introduction and establishment, invasive plants modify the soil microbial communities and soil biochemistry affecting bioremediation efforts and future plant communities. Here, we used tag-encoded FLX amplicon 454 pyrosequencing (TEFAP) to characterize the bacterial and fungal community diversity in the rhizosphere of Berberis thunbergii DC. (Japanese barberry) from invasive stands in coastal Maine to investigate effects of soil type, soil chemistry and surrounding plant cover on the soil microbial community structure. Acidobacteria, Actinobacteria, Proteobacteria and Verrucomicrobia were the dominant bacterial phyla, whereas fungal communities were comprised mostly of Ascomycota and Basidiomycota phyla members, including Agaricomycetes and Sordariomycetes. Bulk soil chemistry had more effect on the bacterial community structure than the fungal community. An effect of geographic location was apparent in the rhizosphere microbial communities, yet it was less significant than the effect of surrounding plant cover. These data demonstrate a high degree of spatial variation in the rhizosphere microbial communities of Japanese barberry with apparent effects of soil chemistry, location and canopy cover on the microbial community structure., (© 2013 John Wiley & Sons Ltd.)

The sea slug Elysia chlorotica offers a unique opportunity to study the evolution of a novel function (photosynthesis) in a complex multicellular host. Elysia chlorotica harvests plastids (absent of nuclei) from its heterokont algal prey, Vaucheria litorea. The "stolen" plastids are maintained for several months in cells of the digestive tract and are essential for animal development. The basis of long-term maintenance of photosynthesis in this sea slug was thought to be explained by extensive horizontal gene transfer (HGT) from the nucleus of the alga to the animal nucleus, followed by expression of algal genes in the gut to provide essential plastid-destined proteins. Early studies of target genes and proteins supported the HGT hypothesis, but more recent genome-wide data provide conflicting results. Here, we generated significant genome data from the E. chlorotica germ line (egg DNA) and from V. litorea to test the HGT hypothesis. Our comprehensive analyses fail to provide evidence for alga-derived HGT into the germ line of the sea slug. Polymerase chain reaction analyses of genomic DNA and cDNA from different individual E. chlorotica suggest, however, that algal nuclear genes (or gene fragments) are present in the adult slug. We suggest that these nucleic acids may derive from and/or reside in extrachromosomal DNAs that are made available to the animal through contact with the alga. These data resolve a long-standing issue and suggest that HGT is not the primary reason underlying long-term maintenance of photosynthesis in E. chlorotica. Therefore, sea slug photosynthesis is sustained in as yet unexplained ways that do not appear to endanger the animal germ line through the introduction of dozens of foreign genes.

The molluscan sea slug Elysia chlorotica is best known for its obligate endosymbiosis with chloroplasts (= kleptoplasty) from its algal prey Vaucheria litorea and its ability to sustain itself photoautotrophically for several months. This unusual photosynthetic sea slug also harbors an array of undescribed bacteria, which may contribute to the long-term success of the symbiosis. Here, we utilized 16S rDNA-based metagenomic analyses to characterize the microbial diversity associated with two populations of E. chlorotica from Halifax, Nova Scotia, Canada, and from Martha's Vineyard, Massachusetts, USA. Animals were examined immediately after collection from their native environments, after being starved of their algal prey for several months, and after being bred in the laboratory (second-generation sea slugs) to characterize the effect of varying environmental and culturing conditions on the associated bacteria. Additionally, the microbiome of the algal prey, laboratory-cultured V. litorea, was analyzed to determine whether the laboratory-bred sea slugs obtained bacteria from their algal food source during development. Bacterial profiles varied between populations and among all conditions except for the F2 laboratory-bred samples, which were similar in diversity and abundance, but not to the algal microbiome. Alpha-, beta-, and gamma-proteobacteria dominated all of the samples along with Actinobacteria, Bacilli, Flavobacteria, and Sphingobacteria. Bacteria capable of polysaccharide digestion and photosynthesis, as well as putative nitrogen fixation, vitamin B(12) production, and natural product biosynthesis were associated with the sea slug and algal samples.

Photosynthesis is composed of tightly coupled reactions requiring finely tuned nucleocytosolic-plastid interaction. Herein, we examined the influence of light on select photosynthetic gene expression and enzyme activity in the plastid-containing mollusk (sea slug) Elysia chlorotica and its heterokont algal prey Vaucheria litorea C. Agardh. Transcript levels of nuclear photosynthetic genes (psbO and prk) were significantly lower in E. chlorotica compared with V. litorea, whereas plastid photosynthesis genes (psaA and rbcL) were more comparable, although still lower in the animal. None of the genes responded similarly to changes in light conditions over a 24 h period in the sea slug compared with the alga. Activity of the nuclear-encoded photosynthetic enzyme phosphoribulokinase (PRK) exhibited redox regulation in vitro in crude extracts of both organisms sequentially treated with oxidizing and reducing agents. However, PRK was differentially affected in vivo by redox and light versus dark treatment in V. litorea, but not in E. chlorotica. Overall, these results support the active transcription of algal nuclear and plastid genes in E. chlorotica, as well as sustained activity of a nuclear-encoded plastid enzyme, even after several months of starvation (absence of algal prey). The apparent absence of tight transcriptional regulation and redox control suggests that essential nuclear-encoded regulatory factors in V. litorea are probably not present in the sea slug. These findings are discussed relative to light regulation of photosynthetic gene expression in the green and red algal lineages and in the context of the sea slug/algal plastid kleptoplastic association., (© 2012 Phycological Society of America.)

Symbiotic animals containing green photobionts challenge the common perception that only plants are capable of capturing the sun's rays and converting them into biological energy through photoautotrophic CO(2) fixation (photosynthesis). 'Solar-powered' sacoglossan molluscs, or sea slugs, have taken this type of symbiotic association one step further by solely harboring the photosynthetic organelle, the plastid (=chloroplast). One such sea slug, Elysia chlorotica, lives as a 'plant' when provided with only light and air as a result of acquiring plastids during feeding on its algal prey Vaucheria litorea. The captured plastids (kleptoplasts) are retained intracellularly in cells lining the digestive diverticula of the sea slug, a phenomenon sometimes referred to as kleptoplasty. Photosynthesis by the plastids provides E. chlorotica with energy and fixed carbon for its entire lifespan of ~10 months. The plastids are not transmitted vertically (i.e. are absent in eggs) and do not undergo division in the sea slug. However, de novo protein synthesis continues, including plastid- and nuclear-encoded plastid-targeted proteins, despite the apparent absence of algal nuclei. Here we discuss current data and provide hypotheses to explain how long-term photosynthetic activity is maintained by the kleptoplasts. This fascinating 'green animal' provides a unique model to study the evolution of photosynthesis in a multicellular heterotrophic organism.

Phosphoribulokinase (PRK), a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction (Calvin) cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc (sea slug) Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclear-encoded proteins that are directed to complex (i.e. four membrane-bound) algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V. litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V. litorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.

The sea slug Elysia chlorotica acquires plastids by ingestion of its algal food source Vaucheria litorea. Organelles are sequestered in the mollusc's digestive epithelium, where they photosynthesize for months in the absence of algal nucleocytoplasm. This is perplexing because plastid metabolism depends on the nuclear genome for >90% of the needed proteins. Two possible explanations for the persistence of photosynthesis in the sea slug are (i) the ability of V. litorea plastids to retain genetic autonomy and/or (ii) more likely, the mollusc provides the essential plastid proteins. Under the latter scenario, genes supporting photosynthesis have been acquired by the animal via horizontal gene transfer and the encoded proteins are retargeted to the plastid. We sequenced the plastid genome and confirmed that it lacks the full complement of genes required for photosynthesis. In support of the second scenario, we demonstrated that a nuclear gene of oxygenic photosynthesis, psbO, is expressed in the sea slug and has integrated into the germline. The source of psbO in the sea slug is V. litorea because this sequence is identical from the predator and prey genomes. Evidence that the transferred gene has integrated into sea slug nuclear DNA comes from the finding of a highly diverged psbO 3' flanking sequence in the algal and mollusc nuclear homologues and gene absence from the mitochondrial genome of E. chlorotica. We demonstrate that foreign organelle retention generates metabolic novelty ("green animals") and is explained by anastomosis of distinct branches of the tree of life driven by predation and horizontal gene transfer.

To analyze quantitative trait loci (QTLs) affecting flooding tolerance and other physiological and morphological traits in Echinochloa crus-galli, a restriction fragment length polymorphism (RFLP) map was constructed using 55 plants of the F(2) population ( E. crus-galli var. praticola x E. crus-galli var. formosensis). One hundred forty-one loci formed 41 linkage groups. The total map size was 1,468 cM and the average size of linkage groups was 35.8 cM. The average distance between markers was 14.7 cM and the range was 0-37.2 cM. Early comparisons to the genetic maps of other taxa suggest appreciable synteny with buffelgrass ( Pennisetum spp.) and sorghum ( Sorghum spp.). One hundred ninty-one F(2) plants were used to analyze QTLs of flooding tolerance, plant morphology, heading date, number of leaves, and plant height. For flooding tolerance, two QTLs were detected and one was mapped on linkage group 24. Other traits, including plant morphology, heading date, number of leaves, and plant height were highly correlated. Three genomic regions accounted for most of the mapped QTLs, each explaining 2-4 of the significant marker-trait associations. The high observed correlation between the traits appears to result from QTLs with a large contribution to the phenotypic variance at the same or nearby locations.

To compare the regulation of anaerobic metabolism during germination in anoxia-tolerant and intolerant plants, enzymes associated with anaerobic metabolism such as sucrose synthase, aldolase, enolase, pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH) were assayed in two varieties of Echinochloa crus-galli, formosensis (tolerant) and praticola (intolerant). The initial and intervening enzymes of the pathway (sucrose synthase and aldolase) and enzymes in the last part of the pathway (PDC, ADH and ALDH) revealed similar changing patterns in activities during germination. This implies that each group of enzymes may be controlled by an identical regulatory mechanism. During anoxia, activities of all enzymes increased 1.5-30-fold in both varieties compared to their activities under aerobic conditions. Activities of sucrose synthase, enolase and ADH exhibited the same induction patterns under anoxia in formosensis and praticola. However, the activities of aldolase, ALDH and PDC were more strongly induced in formosensis under anoxia (1.2-2-fold) than in praticola. These enzymes were also assayed in F(3) families which varied in their anaerobic germinability. For PDC, activities under anoxia in anoxia-tolerant families were similar to those of an anoxia-intolerant family during the whole period although the family did not exhibit anaerobic germinability. This suggests that there is no correlation between PDC activity and anaerobic germinability. For ALDH, activities were more strongly induced under anoxia in anoxia-tolerant families than in anoxia-intolerant families, a trend also exhibited by the parents. This indicates that ALDH may play a role in detoxifying acetaldehyde formed through alcoholic fermentation during anaerobic germination.

A marine sea slug, Elysia chlorotica, has acquired the ability to carry out photosynthesis as a result of forming an intracellular symbiotic association with chloroplasts of the chromophytic alga, Vaucheria litorea. The symbiont chloroplasts (kleptoplasts) are functional, i.e. they evolve oxygen and fix CO(2) and actively transcribe and translate proteins for several months in the sea slug cytosol. Considering the dependency of plastid function on nuclear genes, the level of kleptoplast activity observed in the animal cell is quite remarkable. Possible factors contributing to this long-lasting functional association that are considered here include: the presence of an algal nuclear genome in the sea slug, autonomous chloroplasts, unusual chloroplast/protein stability, re-directing of animal proteins to the kleptoplast, and lateral gene transfer. Based on our current understanding, the acquisition and incorporation of intact algal plastids by E. chlorotica is aided by the robustness of the plastids and the long-term functional activity of the kleptoplasts appears to be supported by both plastid and protein stability and contributions from the sea slug.

Early in its life cycle, the marine mollusc Elysia chlorotica Gould forms an intracellular endosymbiotic association with chloroplasts of the chromophytic alga Vaucheria litorea C. Agardh. As a result, the dark green sea slug can be sustained in culture solely by photoautotrophic CO(2) fixation for at least 9 months if provided with only light and a source of CO(2). Here we demonstrate that the sea slug symbiont chloroplasts maintain photosynthetic oxygen evolution and electron transport activity through photosystems I and II for several months in the absence of any external algal food supply. This activity is correlated to the maintenance of functional levels of chloroplast-encoded photosystem proteins, due in part at least to de novo protein synthesis of chloroplast proteins in the sea slug. Levels of at least one putative algal nuclear encoded protein, a light-harvesting complex protein homolog, were also maintained throughout the 9-month culture period. The chloroplast genome of V. litorea was found to be 119.1 kb, similar to that of other chromophytic algae. Southern analysis and polymerase chain reaction did not detect an algal nuclear genome in the slug, in agreement with earlier microscopic observations. Therefore, the maintenance of photosynthetic activity in the captured chloroplasts is regulated solely by the algal chloroplast and animal nuclear genomes.

In a few well-known cases, animal population dynamics are regulated by cyclical infections of protists, bacteria, or viruses. In most of these cases, the pathogen persists in the environment, where it continues to infect some percentage of successive generations of the host organism. This persistent re-infection causes a long-lived decline, in either population size or cycle, to a level that depends upon pathogen density and infection level (1-4). We have discovered, on the basis of 9 years of observation, an annual viral expression in Elysia chlorotica, an ascoglossan sea slug, that coincides with the yearly, synchronized death of all the adults in the population. This coincidence of viral expression and mass death is ubiquitous, and it occurs in the laboratory as well as in the field. Our evidence also suggests that the viruses do not re-infect subsequent generations from an external pathogen pool, but are endogenous to the slug. We are led, finally, to the hypothesis that the viruses may be involved in the maintenance of symbiotic chloroplasts within the molluscan cells.

Hexokinase (HXK; EC 2.7.1.1) regulates carbohydrate entry into glycolysis and is known to be a sensor for sugar-responsive gene expression. The effect of abiotic stresses on HXK activity was determined in seedlings of the flood-tolerant plant Echinochloa phyllopogon (Stev.) Koss and the flood-intolerant plant Echinochloa crus-pavonis (H.B.K.) Schult grown aerobically for 5 d before being subjected to anaerobic, chilling, heat, or salt stress. HXK activity was stimulated in shoots of E. phyllopogon only by anaerobic stress. HXK activity was only transiently elevated in E. crus-pavonis shoots during anaerobiosis. In roots of both species, anoxia and chilling stimulated HXK activity. Thus, HXK is not a general stress protein but is specifically induced by anoxia and chilling in E. phyllopogon and E. crus-pavonis. In both species HXK exhibited an optimum pH between 8.5 and 9.0, but the range was extended to pH 7.0 in air-grown E. phyllopogon to 6.5 in N2-grown E. phyllopogon. At physiologically relevant pHs (6.8 and 7.3, N2 and O2 conditions, respectively), N2-grown seedlings retained greater HXK activity at the lower pH. The pH response suggests that in N2-grown seedlings HXK can function in a more acidic environment and that a specific isozyme may be important for regulating glycolytic activity during anaerobic metabolism in E. phyllopogon.

The marine slug Elysia chlorotica (Gould) forms an intracellular symbiosis with photosynthetically active chloroplasts from the chromophytic alga Vaucheria litorea (C. Agardh). This symbiotic association was characterized over a period of 8 months during which E. chlorotica was deprived of V. litorea but provided with light and CO2. The fine structure of the symbiotic chloroplasts remained intact in E. chlorotica even after 8 months of starvation as revealed by electron microscopy. Southern blot analysis of total DNA from E. chlorotica indicated that algal genes, i.e., rbcL, rbcS, psaB, psbA, and 16S rRNA are present in the animal. These genes are typically localized to the plastid genome in higher plants and algae except rbcS, which is nuclear-encoded in higher plants and green (chlorophyll a/b) algae. Our analysis suggests, however, that similar to the few other chromophytes (chlorophyll a/c) examined, rbcS is chloroplast encoded in V. litorea. Levels of psbA transcripts remained constant in E. chlorotica starved for 2 and 3 months and then gradually declined over the next 5 months corresponding with senescence of the animal in culture and in nature. The RNA synthesis inhibitor 6-methylpurine reduced the accumulation of psbA transcripts confirming active transcription. In contrast to psbA, levels of 16S rRNA transcripts remained constant throughout the starvation period. The levels of the photosystem II proteins, D1 and CP43, were high at 2 and 4 months of starvation and remained constant at a lower steady-state level after 6 months. In contrast, D2 protein levels, although high at 2 and 4 months, were very low at all other periods of starvation. At 8 months, de novo synthesis of several thylakoid membrane-enriched proteins, including D1, still occurred. To our knowledge, these results represent the first molecular evidence for active transcription and translation of algal chloroplast genes in an animal host and are discussed in relation to the endosymbiotic theory of eukaryote origins.

Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) has been identified as an anaerobic stress protein in Echinochloa oryzoides based on the homology of its internal amino acid sequence with those of enolases from other organisms, by immunological reactivity, and induction of catalytic activity during anaerobic stress. Enolase activity was induced 5-fold in anoxically treated seedlings of three flood-tolerant species (E. oryzoides, Echinochloa phyllopogon, and rice [Oryza sativa L.]) but not in the flood-intolerant species (Echinochloa crus-pavonis). A 540-bp fragment of the enolase gene was amplified by polymerase chain reaction from cDNAs of E. phyllopogon and maize (Zea mays L.) and used to estimate the number of enolase genes and to study the expression of enolase transcripts in E. phyllopogon, E. crus-pavonis, and maize. Southern blot analysis indicated that only one enolase gene is present in either E. phyllopogon or E. crus-pavonis. Three patterns of enolase gene expression were observed in the three species studied. In E. phyllopogon, enolase induction at both the mRNA and enzyme activity levels was sustained at all times with a further induction after 48 h of anoxia. In contrast, enolase was induced in hypoxically treated maize root tips only at the mRNA level. In E. crus-pavonis, enolase mRNA and enzyme activity were induced during hypoxia, but activity was only transiently elevated. These results suggest that enolase expression in maize and E. crus-pavonis during anoxia are similarly regulated at the transcriptional level but differ in posttranslational regulation, whereas enolase is fully induced in E. phyllopogon during anaerobiosis.

Efforts to study the cellular and molecular biology of the symbiotic association between opisthobranch molluscs and algal chloroplasts have been hampered by the copious amounts of mucus produced by the animals. We report for the first time a procedure for isolating total DNA free of contaminating mucilaginous compounds from the mollusc Elysia chlorotica Gould that harbors photosynthetically active chloroplasts from the siphonaceous alga, Vaucheria litorea C. Agardh. This method involves an initial extraction of fresh or freeze-dried Elysia tissue in absolute ethanol and differential processing of the resultant two-phase pellet. Final purification by CsCl-gradient centrifugation produces high molecular weight DNA suitable for molecular analysis.

Echinochloa species differ in their ability to germinate and grow in the absence of oxygen. Seeds of Echinochloa crus-pavonis (H.B.K.) Schult do not germinate under anoxia but remain viable for extended periods (at least 30 d) when incubated in an anaerobic environment. E. crus-pavonis can be induced to germinate and grow in an anaerobic environment if the seeds are first subjected to a short (1-18 h) exposure to aerobic conditions (aerobic priming). Changes in polypeptide patterns (constitutive and de novo synthesized) and protein phosphorylation induced by aerobic priming were investigated. In the absence of aerobic priming protein degradation was not evident under anaerobic conditions, although synthesis of a 20-kD polypeptide was induced. During aerobic priming, however, synthesis of 37- and 55-kD polypeptides was induced and persisted upon return of the seeds to anoxia. Furthermore, phosphorylation of two 18-kD polypeptides was observed only in those seeds that were labeled with 32PO4 during the aerobic priming period. Subsequent chasing in an anaerobic environment resulted in a decrease in phosphorylation of these polypeptides. Likewise, phosphorylation of the 18-kD polypeptides was not observed if the seeds were labeled in an anaerobic atmosphere. These results suggest that the regulated induction of the 20-, 37-, and 55- kD polypeptides may be important for anaerobic germination and growth of E. crus-pavonis and that the specific phosphorylation of the 18-kD polypeptides may be a factor in regulating this induction.

Anaerobic stress resulted in a change in the protein accumulation patterns in shoots of several Echinochloa (barnyard grass) species and Oryza sativa (L.) (rice) as resolved by two-dimensional gel electrophoresis. Of the six Echinochloa species investigated, E. phyllopogon (Stev.) Koss, E. muricata (Beauv.) Fern, E. oryzoides (Ard.) Fritsch Clayton, and E. crus-galli (L.) Beauv. are tolerant of anaerobiosis and germinate in the absence of oxygen, as does rice. In contrast, E. crus-pavonis (H.B.K.) Schult and E. colonum (L.) Link are intolerant and do not germinate without oxygen. Computer analysis of the protein patterns from the four tolerant species and rice indicated that the anaerobic response is of five classes: class 1 proteins, enhanced under anaerobiosis (9 to 13 polypeptides ranging from 16-68 kD); class 2 proteins, unique to anaerobiosis (1 to 5 polypeptides ranging from 17-69 kD); class 3 proteins, remained constant under aerobiosis and anaerobiosis; class 4 proteins, prominent only in air and repressed under anoxia (3 to 7 polypeptides ranging from 19-45 kD); and class 5 proteins, unique to aerobiosis (1 to 4 polypeptides ranging from 18-63 kD). In the intolerant species, E. colonum and E. crus-pavonis, no polypeptides were enhanced or repressed under anoxia (class 1 and class 4, respectively), whereas in the tolerant Echinochloa species and rice, a total of at least 9 to 13 anaerobic stress proteins and 4 to 7 "aerobic" proteins were noted. Immunoblotting identified two of the major anaerobic stress proteins as fructose-1,6-bisphosphate aldolase and pyruvate decarboxylase. Based on the differential response of the intolerant species to anaerobiosis, we suggest that another set of genes, whose products may not necessarily be among the major anaerobic stress polypeptides, might confer tolerance in Echinochloa under prolonged anaerobic stress.

Exposure to oxygen deficits is more widespread in biological systems than is commonly believed. Until recently, the general perception of anaerobic metabolism was often limited to the induction of alcoholic or lactic acid fermentation as the sole biochemical response to hypoxia/anoxia. Developments in the physiology, biochemistry, and molecular biology of anaerobic responses in invertebrates, lower plants, and higher plants have demonstrated that, depending upon the species, anaerobic metabolism may encompass much more than simple glycolytic metabolism. Here, recent progress in elucidating the mechanism(s) determining tolerance versus intolerance to anaerobic environments in higher plants is discussed, drawing most heavily on experimental systems using seeds or seedlings.

Echinochloa crus-galli L. Beauv., a rice-field weed, can germinate and grow for extended periods of time in an anaerobic environment. Compared to pea, which does not germinate under anaerobiosis, the evolution of CO(2) in Echinochloa and rice is lower and the peak rate of CO(2) evolution is delayed when germinated without oxygen. The plants studied also differ with respect to their respiration ratio ([CO(2)] N(2)/[CO(2)] air) and metabolism used during the early stages of germination. Echinochloa does not increase its glycolytic rate under anaerobiosis, whereas pentose phosphate pathway activity appears to increase during the first 40 to 50 hours of germination.Based on its response to metabolic inhibitors (NaF, dinitrophenol, and malonate), anaerobic metabolism in Echinochloa proceeds primarily through glycolysis, with partial operation of the tricarboxylic acid cycle and little or no oxidative phosphorylation. Also, Echinochloa is sensitive to CN during aerobic germination, whereas rice appears to be able to shift to CN-insensitive electron transport. Finally, the effectiveness of cyanide and azide on inhibiting germination of Echinochloa in N(2), but not CO, suggests that cytochrome oxidase is not used to reoxidize pyridine nucleotides in the absence of oxygen. The possible existence of an alternate electron acceptor is discussed.

Echinochloa crus-galli, a problem weed in rice fields, has the rare ability to germinate and to grow in a totally oxygen-free environment. After 7 days growth in the light or dark under N(2), E. crus-galli var. oryzicola produces a 2- to 3-centimeter nonpigmented shoot.Ethanol, malate, and lactate were measured in seeds germinated under N(2) and air, and compared with changes in the activities of alcohol dehydrogenase, malate dehydrogenase, NADP-malic enzyme, and phosphoenolpyruvate carboxylase. During a 7-day time course, ethanol levels increased 661 micromoles per gram dry weight under N(2), with no increase under air. Alcohol dehydrogenase activity increased 5.5 micromoles per gram dry weight per minute in N(2) compared to 1.0 in air. Corresponding increases for lactate were 7.9 micromoles per gram dry weight under N(2) and 2.7 under air, and for malate, 5.3 micromoles per gram dry weight under N(2) and 0.4 under air.Although 85% of the ethanol produced by the seedlings was found in the external solution under N(2), the seeds still contained 90 times more ethanol under anaerobic conditions than under air. No phosphoenolpyruvate carboxykinase activity was detected and phosphoenolpyruvate carboxylase activity was 10 times less under N(2) than air. Malic enzyme activity increased 5-fold under anaerobic conditions, comparable to the change under aerobic conditions. Proposed adaptive mechanisms of flood-tolerant species to anaerobiosis are discussed.

3-Phosphoglycerate (PGA)-dependent O(2) evolution by mesophyll chloroplasts of the C(4) plant, Digitaria sanguinalis L. Scop. (crabgrass), was inhibited by micromolar levels of 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). As little as 1.8 micromolar DIDS added to the assay medium (containing 0.7 millimolar PGA) resulted in 80 to 100% inhibition of O(2) evolution. The extent of inhibition of O(2) evolution observed was dependent on various factors including: pH, concentration of DIDS to relative chlorophyll, concentration of PGA, and the time of addition of DIDS to the chloroplasts relative to addition of PGA.Preincubation of crabgrass chloroplasts with micromolar levels of DIDS, followed by washing to remove any nonirreversibly bound DIDS, inhibited PGA-dependent O(2) evolution. Protection against this inhibition was afforded by preincubating the chloroplasts with various substrates before adding DIDS. For example, if the chloroplasts were first incubated with 8.3 millimolar PGA, phosphoenolpyruvate (PEP) or inorganic phosphate before adding 42 micromolar DIDS, the percentage of inhibition was decreased from 100% (without any substrate) to 0, 54, and 67%, respectively. 2-Phosphoglycerate caused a slight decrease in the inhibition (about 10%) and glucose-6-phosphate had no protective effect. If the chloroplasts were pretreated with DIDS initially, the inhibition could not be overcome by PGA, suggesting that DIDS acts as an irreversible inhibitor. Micromolar levels of DIDS also inhibited PGA dependent O(2) evolution by isolated chloroplasts of the C(3) plant barley. As with crabgrass, preincubation with PGA or inorganic phosphate resulted in a decrease in the DIDS inhibition, but PEP was very ineffective compared to the C(4) chloroplasts.Oxalacetate-dependent O(2) evolution and its stimulation by the uncoupler, NH(4)Cl, were unaffected by the addition of DIDS to crabgrass mesophyll chloroplasts. Furthermore, preincubation of the chloroplasts with DIDS (up to 65 micromolar) had no inhibitory effect on the extractable activity of NADP glyceraldehyde-3-P dehydrogenase and phosphoglycerate kinase. Inhibition by DIDS was interpreted to be at the substrate binding site of the phosphate translocator. The data further suggest that in C(4) crabgrass chloroplasts, PEP is transported on a carrier which also transports PGA.

The usefulness of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C3 plant) and maize (Zea mays L.) (C4 plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [(3)H2]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, Mr, ∼30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [(3)H2]DIDS. However, when broken chloroplasts were incubated with [(3)H2]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [(3)H2]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the ∼30-kDa protein - the presumptive phosphate translocator - in C3 and C4 chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the ∼30-kDa protein in the inner membrane of the envelope.

Tolerance to ethanol and the ability to metabolize key intermediary substrates under anaerobiosis were studied in Echinochloa crus-galli (L.) Beauv. var oryzicola seeds to further characterize the mechanisms which enable it to germinate and grow without O(2).Our results indicate that E. crus-galli var oryzicola possesses an inherently high tolerance to ethanol and is able to metabolize low levels of ethanol in the absence of O(2). Concentrations of ethanol 45-fold greater than endogenous levels did not prove toxic to germinating seeds.Five-day anaerobically grown seedlings of E. crus-galli var oryzicola metabolized added [(14)C]sucrose primarily to CO(2) and ethanol. Of the soluble compounds labeled, the phosphorylated intermediates of glycolysis and the oxidative pentose phosphate pathway predominated more under anaerobiosis than in air. In addition, organic acids and lipids were labeled from [(14)C]sucrose, the latter indicating that metabolism of carbohydrate via acetyl-CoA occurred in the absence of O(2). Lipids were also labeled when seeds were supplied with [(14)C]ethanol or [(14)C]acetate. Labeling experiments using the above compounds plus [(14)C]NaHCO(3), showed further labeling of organic acids; succinate and citrate being labeled under nitrogen, while fumarate was formed in air.The above metabolic characteristics would allow for the maintenance of an active alcoholic fermentation system which, along with high alcohol dehydrogenase activity, would continue to recycle NAD and result in continued energy production without O(2). In addition, Echinochloa's ability to metabolize carbohydrate intermediates and to synthesize lipids indicates that mechanisms exist for providing the carbon intermediates for biosynthesis, particularly membrane synthesis for growth, even in the absence of O(2).

The initial products of photosynthesis by the C(3) species Flaveria cronquistii, the C(4) species F. trinervia, and the C(3)-C(4) intermediate species F. ramosissima were determined using a pulse-chase technique with (14)CO(2)-(12)CO(2). The intermediate species F. ramosissima incorporated at least 42% of the total soluble (14)C fixed into malate and aspartate after 10 seconds of photosynthesis in (14)CO(2), as compared with 90% for the C(4) species F. trinervia and 5% for the C(3) species F. cronquistii. In both F. ramosissima and F. trinervia, turnover of labeled malate and aspartate occurred during a chase period in (12)CO(2), although the rate of turnover was slower in the intermediate species. Relative to F. cronquistii, F. ramosissima showed a reduced incorporation of radioactivity into serine and glycine during the pulse period. These results indicate that a functional C(4) pathway of photosynthesis is operating in F. ramosissima which can account for its reduced level of photorespiration, and that this species is a true biochemical intermediate between C(3) and C(4) plants.

The effects of phosphoenolpyruvate (PEP), inorganic phosphate (Pi), and ATP on 3-phosphoglycerate (PGA)-dependent O(2) evolution by chloroplasts of Digitaria sanguinalis (L.) Scop. (crabgrass) were evaluated relative to possible mechanisms of PEP transport by the C(4) mesophyll chloroplast. Crude and Percoll purified chloroplast preparations exhibited rates of PGA-dependent O(2) evolution in the range of 90 to 135 micromoles O(2) per milligram chlorophyll per hour, and up to 180 micromoles O(2) per milligram chlorophyll per hour at optimal Pi concentrations (approximately 0.2 millimolar at 9 millimolar PGA). Higher concentrations of Pi were inhibitory. PEP inhibited O(2) evolution (up to 70%) in both chloroplast preparations when the PEP to PGA ratio was high (i.e. 9 millimolar PEP to 0.36 millimolar PGA). Usually no inhibition was seen when the PEP to PGA ratio was less than 2. PEP acted as a competitive inhibitor and, at a concentration of 9 millimolar, increased the apparent K(m) (PGA) from 0.15 to 0.53 millimolar in Percoll purified chloroplasts. A low concentration of PGA and high ratio of PEP to PGA, which are considered unphysiological, were required to detect any inhibition of O(2) evolution by PEP. Similar results were obtained from crude versus Percoll purified preparations. Neither the addition of Pi nor ATP could overcome PEP inhibition. As PEP inhibition was competitive with respect to PGA concentration, and as addition of ATP or Pi could not prevent PEP inhibition of PGA-dependent O(2) evolution, the inhibition was not due to PEP exchange of adenylates or Pi out of the chloroplast. Analysis of the effect of Pi and PEP, separately and in combination, on PGA-dependent O(2) evolution suggests interactions between PEP, Pi, and PGA on the same translocator in the C(4) mesophyll chloroplast. C(3) spinach chloroplasts were also found to be sensitive to PEP, but to a lesser extent than crabgrass chloroplasts. The apparent K(i) values (PEP) were 3 and 21 millimolar for crabgrass and spinach, respectively.

In the polyol producing plant, celery (Apium graveolens L.), mannitol is a major photosynthetic product and a form in which carbohydrate is translocated. Measurements of whole leaf extracts of celery indicated substantial activity of the following enzymes: mannose-6-P reductase, mannose-6-P isomerase, mannitol-1-P phosphatase, and nonreversible glyceraldehyde-3-P dehydrogenase. The activities of these enzymes were either undetectable or very low in the nonpolyol producing plants, Secale cereale L. (rye) and Vigna mungo (L.) Hepper (black gram).Mesophyll protoplasts were enzymically isolated from celery leaves, broken with a Yeda press and the intracellular localization of the above enzymes for mannitol synthesis studied following differential and/or sucrose density gradient centrifugation of the protoplast extract. These data suggested the enzymes involved in mannitol synthesis are exclusively localized in the cytoplasm. Ninety-five to 100% of the activity of these enzymes, along with the cytoplasmic marker enzyme phosphoenolpyruvate carboxylase, was found in the cytosolic fraction.We propose the pathway of photosynthetic carbon flow from triose-P to mannitol in celery occurs via fructose-6-P, mannose-6-P, and mannitol-1-P; these final reactions being catalyzed by the cytoplasmic enzymes, mannose-6-P isomerase, NADPH-dependent mannose-6-P reductase, and mannitol-1-P phosphatase, respectively. The requirement for NADPH may be met via the cytoplasmically located NADP-linked nonreversible glyceraldehyde-3-P dehydrogenase.

Intact chloroplasts were obtained from mesophyll protoplasts isolated from Mesembryanthemum crystallinum in the C3 or Crassulacean acid metabolism (CAM) photosynthetic mode, and examined for the influence of inorganic phosphate (Pi) on aspects of bicarbonate-dependent O2 evolution and CO2 fixation. While the chloroplasts from both modes responded similarly to varying Pi, some features appear typical of chloroplasts from species capable of CAM, including a relatively high capacity for photosynthesis in the absence of Pi, a short induction period, and resistance to inhibition of photosynthesis by high levels of Pi. In the absence of Pi the chloroplasts retained 75-85% of the (14)CO2 fixed and the total export of dihydroxyacetone phosphate was low compared with the rate of photosynthesis. In CAM plants the ability to conduct photosynthesis and retain most of the fixed carbon in the chloroplasts at low external Pi concentrations may enable storage of carbohydrates which are essential for providing a carbon source for the nocturnal synthesis of malic acid. At high external Pi concentrations (e.g. 10 25 mM), the amount of total dihydroxyacetone phosphate exported to the assay medium relative to the rate of photosynthesis was high while the products of (14)CO2 fixation were largely retained in the chloroplasts which indicates starch degradation is occurring at high Pi levels. Starch degradation normally occurs in CAM plants in the dark; high levels of Pi may induce starch degradation in the light which has the effect of limiting export of the immediate products of photosynthesis and thus the degree of Pi inhibition of photosynthesis with the isolated chloroplast.

The phosphate translocator protein of C(3) and C(4) mesophyll chloroplast envelopes was specifically labeled using the anion exchange inhibitor, 1,2-ditritio-1,2-(2,2' -disulfo-4,4' -diisothiocyano) diphenylethane ([(3)H](2)-DIDS). Intact mesophyll chloroplasts were isolated from the C(3) plants, Spinacia oleracea L. (spinach) and Pisum sativum L. (pea), and the C(4) plant, Zea mays L. (corn). Chloroplasts were incubated with 5 to 50 mum [(3)H](2)-DIDS and, in addition, pea chloroplasts were also incubated with pyridoxal phosphate/tritiated sodium borohydride. The chloroplasts were washed, the envelopes isolated and solubilized. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, label from bound [(3)H](2)-DIDS was detected only in the 28- to 30-kilodalton protein (proposed C(3) phosphate translocator) for both C(3) and C(4) chloroplasts, as demonstrated by fluorography. In contrast, when pyridoxal phosphate/tritiated sodium borohydride was used to label pea chloroplasts, radioactivity was detected in several other bands in addition to the 29-kilodalton polypeptide. These findings suggest that DIDS is a much more specific inhibitor than reagents previously employed to study the phosphate translocator and could be used to isolate and characterize the differences in the C(3) and C(4) phosphate translocator protein(s).