Single-cell analysis reveals an active and heterotrophic microbiome in the Guaymas Basin deep subsurface with significant inorganic carbon fixation by heterotrophs.

The marine subsurface is a long-term sink of atmospheric carbon dioxide with significant implications for climate on geologic timescales. Subsurface microbial cells can either enhance or reduce carbon sequestration in the subsurface, depending on their metabolic lifestyle. However, the activity of subsurface microbes is rarely measured. Here, we used nanoscale secondary ion mass spectrometry (nanoSIMS) to quantify anabolic activity in 3,203 individual cells from the thermally altered deep subsurface in the Guaymas Basin, Mexico (3-75 m below the seafloor, 0-14°C). We observed that a large majority of cells were active (83%-100%), although the rates of biomass generation were low, suggesting cellular maintenance rather than doubling. Mean single-cell activity decreased with increasing sediment depth and temperature and was most strongly correlated with porewater sulfate concentrations. Intracommunity heterogeneity in microbial activity decreased with increasing sediment depth and age. Using a dual-isotope labeling approach, we determined that all active cells analyzed were heterotrophic, deriving the majority of their cellular carbon from organic sources. However, we also detected inorganic carbon assimilation in these heterotrophic cells, likely via processes such as anaplerosis, and determined that inorganic carbon contributes at least 5% of the total biomass carbon in heterotrophs in this community. Our results demonstrate that the deep marine biosphere at Guaymas Basin is largely active and contributes to subsurface carbon cycling primarily by not only assimilating organic carbon but also fixing inorganic carbon. Heterotrophic assimilation of inorganic carbon may be a small yet significant and widespread underappreciated source of labile carbon in the global subsurface. IMPORTANCE: The global subsurface is the largest reservoir of microbial life on the planet yet remains poorly characterized. The activity of life in this realm has implications for long-term elemental cycling, particularly of carbon, as well as how life survives in extreme environments. Here, we recovered cells from the deep subsurface of the Guaymas Basin and investigated the level and distribution of microbial activity, the physicochemical drivers of activity, and the relative significance of organic versus inorganic carbon to subsurface biomass. Using a sensitive single-cell assay, we found that the majority of cells are active, that activity is likely driven by the availability of energy, and that although heterotrophy is the dominant metabolism, both organic and inorganic carbon are used to generate biomass. Using a new approach, we quantified inorganic carbon assimilation by heterotrophs and highlighted the importance of this often-overlooked mode of carbon assimilation in the subsurface and beyond.

Reserve mobilization and secondary metabolites during seed germination and seedling establishment of the tree Erythrina velutina (Fabaceae) / Movilización de reservas y metabolitos secundarios durante la germinación de semillas y el establecimiento de plántulas del árbol Erythrina velutina (Fabaceae)

Introduction: The lack of knowledge on seed germination and seedling establishment is a main constraint for the restoration of degraded areas, including the tropical dry forest known as Caatinga. Objective: To assess reserve and secondary metabolite mobilization during seed germination and seedling establishment in Erythina velutina. Methods: We scarified, disinfected, imbibed, sown between towel paper, and incubated seeds under controlled conditions. We hydroponically cultivated seedlings in a greenhouse. We harvested cotyledons at seed imbibition, radicle protrusion, hypocotyl emergence, apical hook formation and expansion of cordiform leaves, first trifoliate leaf, and second trifoliate leaf. Results: Seeds contained approximately 20 % starch, 14.5 % storage proteins, 11.6 % neutral lipids, and 5.7 % non-reducing sugars on a dry weight basis. Soluble sugars were mainly consumed from hypocotyl emergence to apical hook formation, while major reserves were mobilized from apical hook formation to expansion of first trifoliate leaf. Enzymatic activity increased from mid to late seedling establishment, causing the mobilization of starch, oils, and proteins. Terpenoid-derivatives, flavonoids, phenolic acids, and alkaloids were detected. Flavonoids and phenolic acids were present at almost all stages and terpenoid-derivatives disappeared at expansion of cordiform leaves. Conclusion: Soluble sugars support early seedling growth, while starch, oils and proteins are simultaneously mobilized from mid to late establishment by amylases, lipases, and acid proteases. The cotyledons contain secondary metabolites, which may act in seedling defense. High content of reserves and presence of secondary metabolites in the cotyledons could enable E. velutina seedlings endure stress, validating their use in the restoration of degraded areas.

Introducción: La falta de conocimiento sobre la germinación de semillas y el establecimiento de plántulas es una de las principales limitaciones para la restauración de áreas degradadas, incluido el bosque seco tropical conocido como Caatinga. Objetivo: Evaluar la movilización de reservas y metabolitos secundarios durante estas etapas de desarrollo en Erythina velutina. Métodos: Las semillas fueron escarificadas, desinfectadas, embebidas, sembradas entre toallas de papel e incubadas bajo condiciones controladas. Cultivamos las plántulas hidropónicamente en un invernadero. Recolectamos los cotiledones en la imbibición de la semilla, la protrusión de la radícula, la emergencia del hipocótilo, la formación del gancho apical y la expansión de las hojas cordiformes, la primera y segunda hoja trifoliada. Resultados: Las semillas contenían 20 % de almidón, 14.5 % de proteínas de almacenamiento, 11.6 % de lípidos neutros y 5.7 % de azúcares no reductores en peso seco. Los azúcares solubles se consumieron desde la emergencia del hipocótilo hasta la formación del gancho apical. Las principales reservas se movilizaron desde la formación del gancho apical hasta la expansión de la primera hoja trifoliada. La actividad enzimática aumentó desde la mitad hasta el final del establecimiento de las plántulas, movilizando almidón, aceites y proteínas. Se detectaron derivados de terpenoides, flavonoides, ácidos fenólicos y alcaloides. Los flavonoides y los ácidos fenólicos estuvieron en casi todas las etapas y los derivados terpenoides desaparecieron en la expansión de las hojas cordiformes. Conclusión: Los azúcares solubles apoyan el crecimiento temprano de las plántulas; el almidón, los aceites y las proteínas se movilizan simultáneamente desde el establecimiento medio hasta el final por amilasas, lipasas y proteasas ácidas. Los cotiledones contienen metabolitos secundarios, que pueden actuar en la defensa de las plántulas. El alto contenido de reservas y los metabolitos secundarios en los cotiledones podría permitir que las plántulas de E. velutina toleren estrés, validando su uso en la restauración de áreas degradadas.

Drivers of phytoplankton production and community structure in nutrient-poor estuaries receiving terrestrial organic inflow.

The influence of nutrient availability and light conditions on phytoplankton size-structure, nutritional strategy and production was studied in a phosphorus-poor estuary in the northern Baltic Sea receiving humic-rich river water. The relative biomass of mixotrophic nanophytoplankton peaked in spring when heterotrophic bacterial production was high, while autotrophic microphytoplankton had their maximum in summer when primary production displayed highest values. Limiting substance (phosphorus) only showed small temporal variations, and the day light was at saturating levels all through the study period. We also investigated if the phytoplankton taxonomic richness influences the production. Structural equation modelling indicated that an increase of the taxonomic richness during the warm summer combined with slightly higher phosphorus concentration lead to increased resource use efficiency, which in turn caused higher phytoplankton biomass and primary production. Our results suggest that climate warming would lead to higher primary production in northerly shallow coastal areas, which are influenced by humic-rich river run-off from un-disturbed terrestrial systems.

Proteomic analysis of grapevine (Vitis vinifera L.) leaf changes induced by transition to autotrophy and exposure to high light irradiance.

Using a proteomics approach, we evaluated the response of heterotrophic and autotrophic leaves of grapevine when exposed to high light irradiation. From a total of 572 protein spots detected on two-dimensional gels, 143 spots showed significant variation caused by changes in the trophic state. High light treatment caused variation in 90 spots, and 51 spots showed variation caused by the interaction between both factors. Regarding the trophic state of the leaf, most of the proteins detected in the heterotrophic stage decreased in abundance when the leaf reached the autotrophic stage. Major differences induced by high light were detected in autotrophic leaves. In the high-light-treated autotrophic leaves several proteins involved in the oxidative stress response were up-regulated. This pattern was not observed in the high-light-treated heterotrophic leaves. This indicates that in these types of leaves other mechanisms different to the protein antioxidant system are acting to protect young leaves against the excess of light. This also suggests that these protective mechanisms rely on other sets of proteins or non-enzymatic molecules, or that differences in protein dynamics between the heterotrophic and autotrophic stages makes the autotrophic leaves more prone to the accumulation of oxidative stress response proteins. BIOLOGICAL SIGNIFICANCE: Transition from a heterotrophic to an autotrophic state is a key period during which the anatomical, physiological and molecular characteristics of a leaf are defined. In many aspects the right functioning of a leaf at its mature stage depends on the conditions under what this transition occurs. This because apart of the genetic control, environmental factors like mineral nutrition, temperature, water supply, light etc. are also important in its control. Many anatomical and physiological changes have been described in several plant species, however in grapevine molecular data regarding changes triggered by this transition or by light stress are still scarce. In this study, we identify that the transition from heterotrophic to autotrophic state in grapevine triggers major changes in the leaf proteome, which are mainly related to processes such as protein synthesis, protein folding and degradation, photosynthesis and chloroplast development. With the exception of proteins involved in carbon fixation, that increased in abundance, most of the proteins detected during the heterotrophic stage decreased in abundance when the leaf reached its autotrophic stage. This is most likely because leaves have reached their full size and from now they have to work as a carbon source for sink organs located in other parts of the plant. Despite the potential control of this transition by light, to date, no studies using a proteomics approach have been conducted to gain a broader view of the effects of short-term high light stress. Our results indicate that short-term high light exposure has a major impact on the proteome of the autotrophic leaves, and trigger a differential accumulation of several proteins involved in the oxidative stress response. Surprisingly, heterotrophic leaves do not display this pattern which can be attributed to a lower sensitivity of these leaves to high light stimulus. In fact we discovered that heterotrophic leaves are more tolerant to light stress than autotrophic leaves. This finding is of high biological significance because it helps to understand how young leaves are able to evolve to autotrophy in areas where high light intensities are predominant. This also reveals in this type of leaves the existence of alternative mechanisms to address this stressful condition. These observations provide new insights into the molecular changes occurring during transition of leaves to autotrophy particularly when this transition occurs under high light intensities. This for example occurs during the springtime when the grapevine buds burst and the young leaves are suddenly exposed to high light intensities.

Nutrient budgets (C, N and P) and trophic dynamicsof a Brazilian tropical estuary: Barra das Jangadas

This paper focuses on the nutrient dynamics of a tropical estuary on the northeastern Brazilian coast, studied using the LOICZ biogeochemical budgeting protocol. We describe the methodology and assumptions underlying this model. Input data (monthly for rainfall, evaporation, river discharge, and concentrations of salt, phosphorus and nitrogen) were obtained during field campaigns in the Barra das Jangadas Estuary (BJE) over a 5 years period (1999 to 2003). Mass balance results indicate large inputs of nutrients to the system. The model shows that the seasonal variation of the Net Ecosystem Metabolism (NEM) indicates that the system passes from a stage of organic matter liquid production and mineralization during the dry season (-0.5 mmoles C m-2 d-1) to liquid mineralization during the rainy season (-19 mmoles C m-2 d-1). We suggest that the system varies slightly between autotrophy and heterotrophy during the year due to the rainfall regime, human activities in the basin (density population and sugarcane plantations), and associated DIP riverine loads. High per capita loads of N and P indicate a high population density and high runoff. The application of flux balance modeling was useful to understand the nutrient dynamics of this typical small tropical estuary.

Este trabalho se focalizou na dinâmica de nutrientes de um estuário tropical na costa nordeste brasileira, usando o protocolo LOICZ de balanços biogeoquímicos. Nós descrevemos a metodologia e os pressupostos subjacentes a este modelo. Os dados de entrada (precipitação e evaporação mensal, vazão do rio, e as concentrações de sal, fósforo e nitrogênio) foram obtidos durante as campanhas de campo no estuário de Barra das Jangadas - Brasil durante um período de 5 anos (1999 a 2003). Os resultados indicam grandes entradas de nutrientes ao sistema. O modelo mostrou que a variação sazonal do Metabolismo do Ecosistema (NEM) indica que o sistema passa de uma fase de produção de líquido da matéria orgânica, durante a estação seca (-0,5 mmoles C m-2 d-1) para uma mineralização líquida durante a estação chuvosa (-19 mmol m-2 C d-1). Sugerimos que o sistema varia ligeiramente entre autotrófica e heterotrófica durante o ano, devido ao regime de chuvas, as atividades antrópicas na bacia (densidade populacional e as plantações de cana de açúcar), e as cargas ribeirinhas de DIP associadas. A alta carga de N e P per capita, indica uma alta densidade populacional e um alto runoff. A aplicação da modelagem de balanço de fluxos foi útil para o entendimento da dinâmica de nutrientes em um pequeno estuário tipicamente tropical.

Sacarose e período de cultivo in vitro na aclimatização ex vitro de ginseng brasileiro (Pfaffia glomerata Spreng. Pedersen)

Pfaffia glomerata (Spreng.) Pedersen is an extensively used plant in popular medicine due to its phytotherapic characteristics. Due to low photosynthetic capacity of plants cultivated in vitro, an extra source of carbohydrates is required to supply their metabolic demands. The growth rate of roots and shoots of P. glomerata are different during the in vitro cultivation. The objectives of this study were to evaluate the effect of sucrose levels and the duration of in vitro growth on ex vitro acclimatization of P. glomerata seedlings. Treatments consisted of a bifactorial combination (5x2) of five sucrose levels (15, 30, 45, 60 and 75g L-1) and two periods of in vitro cultivation (25 and 32 days after inoculation). In the in vitro cultivation, the experimental unit consisted of a test tube containing 10mL of MS medium and a nodal segment, from in vitro cultivated plants, of 1,0cm in length without leaves. The acclimatization scheme consisted of ex vitro cultivation by four successive phases: (i) opening of the test tube and subsequent cultivation in growth room for three days; (ii) seedlings subcultivation to Plantmax substrate and cultivation in growth room for 21 days; (iii) seedlings transfer to partially shady natural environment for 24 days; and (iv) seedling subcultivation to soil under field condition. The greatest plant growth by increasing the sucrose availability (levels among 45 and 60g L-1) during the in vitro growth contributed to acclimatization. Regardless of duration of the in vitro growth, the seedlings obtained and efficient acclimatization. The acclimatization procedure was 100% efficient on seedling production of P. glomerata.

Pfaffia glomerata (Spreng.) Pedersen é uma planta extensivamente usada na medicina popular em decorrência de possuir propriedades fitoterápicas. Devido à sua baixa capacidade fotossintética, as plantas cultivadas in vitro requerem uma fonte extra de carboidratos para suprir suas necessidades metabólicas. O tempo de cultivo in vitro influencia as taxas de crescimento das raízes e da parte aérea de P. glomerata. Este trabalho teve como objetivos avaliar os efeitos da sacarose e do período de cultivo in vitro na aclimatização ex vitro de plântulas de P. glomerata. Os tratamentos consistiram de uma combinação bifatorial (5x2) entre cinco concentrações de sacarose (15, 30, 45, 60 e 75g L-1) e dois períodos de cultivo in vitro (25 e 32 dias após a inoculação). No cultivo in vitro, a parcela experimental consistiu de um tubo de ensaio contendo 10mL de meio MS e um segmento nodal, obtidos de plântulas mantidas in vitro, de 1,0cm de comprimento e sem folhas. O procedimento de aclimatização consistiu de quatro fases sucessivas de cultivo ex vitro: (i) abertura dos tubos de ensaio e exposição das plântulas ao ambiente de câmara climatizada por três dias; (ii) transplantio para substrato Plantmax e cultivo em condições de câmara climatizada por 21 dias; (iii) transferência das mudas para ambiente natural parcialmente sombreado, com duração de 24 dias; e (iv) transplantio para solo em condições de cultivo a campo. O maior crescimento das plantas obtido pelo aumento da disponibilidade de sacarose (concentrações entre 45 e 60g L-1) no cultivo in vitro contribuiu para a aclimatização. Independente do período de retirada das plantas do cultivo in vitro, as mudas obtiveram adequada aclimatização. O procedimento de aclimatização foi 100% eficiente na produção de mudas de P. glomerata.

Sacarose e período de cultivo in vitro na aclimatização ex vitro de ginseng brasileiro (Pfaffia glomerata Spreng. Pedersen)

Pfaffia glomerata (Spreng.) Pedersen is an extensively used plant in popular medicine due to its phytotherapic characteristics. Due to low photosynthetic capacity of plants cultivated in vitro, an extra source of carbohydrates is required to supply their metabolic demands. The growth rate of roots and shoots of P. glomerata are different during the in vitro cultivation. The objectives of this study were to evaluate the effect of sucrose levels and the duration of in vitro growth on ex vitro acclimatization of P. glomerata seedlings. Treatments consisted of a bifactorial combination (5x2) of five sucrose levels (15, 30, 45, 60 and 75g L-1) and two periods of in vitro cultivation (25 and 32 days after inoculation). In the in vitro cultivation, the experimental unit consisted of a test tube containing 10mL of MS medium and a nodal segment, from in vitro cultivated plants, of 1,0cm in length without leaves. The acclimatization scheme consisted of ex vitro cultivation by four successive phases: (i) opening of the test tube and subsequent cultivation in growth room for three days; (ii) seedlings subcultivation to Plantmax substrate and cultivation in growth room for 21 days; (iii) seedlings transfer to partially shady natural environment for 24 days; and (iv) seedling subcultivation to soil under field condition. The greatest plant growth by increasing the sucrose availability (levels among 45 and 60g L-1) during the in vitro growth contributed to acclimatization. Regardless of duration of the in vitro growth, the seedlings obtained and efficient acclimatization. The acclimatization procedure was 100% efficient on seedling production of P. glomerata.

Pfaffia glomerata (Spreng.) Pedersen é uma planta extensivamente usada na medicina popular em decorrência de possuir propriedades fitoterápicas. Devido à sua baixa capacidade fotossintética, as plantas cultivadas in vitro requerem uma fonte extra de carboidratos para suprir suas necessidades metabólicas. O tempo de cultivo in vitro influencia as taxas de crescimento das raízes e da parte aérea de P. glomerata. Este trabalho teve como objetivos avaliar os efeitos da sacarose e do período de cultivo in vitro na aclimatização ex vitro de plântulas de P. glomerata. Os tratamentos consistiram de uma combinação bifatorial (5x2) entre cinco concentrações de sacarose (15, 30, 45, 60 e 75g L-1) e dois períodos de cultivo in vitro (25 e 32 dias após a inoculação). No cultivo in vitro, a parcela experimental consistiu de um tubo de ensaio contendo 10mL de meio MS e um segmento nodal, obtidos de plântulas mantidas in vitro, de 1,0cm de comprimento e sem folhas. O procedimento de aclimatização consistiu de quatro fases sucessivas de cultivo ex vitro: (i) abertura dos tubos de ensaio e exposição das plântulas ao ambiente de câmara climatizada por três dias; (ii) transplantio para substrato Plantmax e cultivo em condições de câmara climatizada por 21 dias; (iii) transferência das mudas para ambiente natural parcialmente sombreado, com duração de 24 dias; e (iv) transplantio para solo em condições de cultivo a campo. O maior crescimento das plantas obtido pelo aumento da disponibilidade de sacarose (concentrações entre 45 e 60g L-1) no cultivo in vitro contribuiu para a aclimatização. Independente do período de retirada das plantas do cultivo in vitro, as mudas obtiveram adequada aclimatização. O procedimento de aclimatização foi 100% eficiente na produção de mudas de P. glomerata.